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United States Patent |
5,270,171
|
Cercek
,   et al.
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December 14, 1993
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Cancer-associated SCM-recognition factor, preparation and method of use
Abstract
A cancer recognition factor (SCM factor) useful in the performance of the
structuredness of the cytoplasmic matrix (SCM) test has been isolated,
purified to substantial homogeneity, and characterized, and methods for
its use have been described. The factor is a peptide of at least 9 amino
acid residues including a core sequence of 9 amino acid residues having an
amphipathicity profile substantially equivalent to that of the sequence
F-L-M-I-D-Q-N-T-K and produces at least a 10 percent decrease in the
intracellular fluorescence polarization value of SCM-responding
lymphocytes from donors afflicted with cancer. A synthetic SCM factor
representing a consensus sequence of
M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K is fully active.
Antibodies specific for SCM factor are useful in immunoassays that can
detect the factor, including detection in cancer cells grown in vitro. The
SCM factor is useful for screening of blood samples and other body fluids
or cell aspirates for the presence of malignancy in the donor. The
multiple action spectrum of the SCM factor including cancer proliferation
and invasion promotion, as well as inhibition of the host's immune defense
mechanisms and synthesis of SCM factor by cancer cells, represents a novel
target for cancer management. Methods for reducing in vivo activity of the
SCM factor, such as dialysis or antibody neutralization, can also be
useful in the management of cancer.
Inventors:
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Cercek; Boris (4318 Camphor Ave., Yorba Linda, CA 92686);
Cercek; Lea (4318 Camphor Ave., Yorba Linda, CA 92686)
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Appl. No.:
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539686 |
Filed:
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June 18, 1990 |
Current U.S. Class: |
435/29; 436/811; 436/813; 530/324; 530/325; 530/326; 530/327; 530/328; 530/350; 530/380 |
Intern'l Class: |
C12Q 001/02; A61K 037/02; A61K 035/14; A61K 037/04; C07K 005/00; C07K 007/00; C07K 015/00; C07K 017/00; C07K /; C07K 013/00 |
Field of Search: |
435/220,183,29
436/813,811
530/328,327,326,325,324,350,380
|
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WO86/03007 | May., 1986 | WO.
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WO90/04785 | May., 1990 | WO.
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445690 | May., 1974 | SU.
| |
06595 | Sep., 1988 | WO.
| |
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|
Primary Examiner: Nucker; Christine M.
Assistant Examiner: Preston; D. R.
Attorney, Agent or Firm: Sheldon & Mak
]>
structuredness of the cytoplasmic matrix (SCM) test has been isolated,
purified to substantial homogeneity, and characterized, and methods for
its use have been described. The factor is a peptide of at least 9 amino
acid residues including a core sequence of 9 amino acid residues having an
amphipathicity profile substantially equivalent to that of the sequence
F-L-M-I-D-Q-N-T-K and produces at least a 10 percent decrease in the
intracellular fluorescence polarization value of SCM-responding
lymphocytes from donors afflicted with cancer. A synthetic SCM factor
representing a consensus sequence of
M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K is fully active.
Antibodies specific for SCM factor are useful in immunoassays that can
detect the factor, including detection in cancer cells grown in vitro. The
SCM factor is useful for screening of blood samples and other body fluids
or cell aspirates for the presence of malignancy in the donor. The
multiple action spectrum of the SCM factor including cancer proliferation
and invasion promotion, as well as inhibition of the host's immune defense
mechanisms and synthesis of SCM factor by cancer cells, represents a novel
target for cancer management. Methods for reducing in vivo activity of the
SCM factor, such as dialysis or antibody neutralization, can also be
useful in the management of cancer.
Parent Case Text
CROSS-REFERENCES
This application is a continuation-in-part of Ser. No. 07/167,007, filed
Mar. 3, 1988, which itself was a continuation-in-part of Ser. No.
07/022,759, filed Mar. 6, 1987, and now abandoned. Both of these prior
patent applications are by Dr. Boris Cercek and Dr. Lea Cercek and are
entitled "General Cancer-Associated SCM-Recognition Factor, Preparation
and Method of Use." This application is also related to prior patent
applications, all by Drs. Boris & Lea Cercek: (1) Ser. No. 06/838,264,
filed Mar. 10, 1986 (now abandoned), and Ser. No. 07/260,928, filed Oct.
21, 1988, a continuation-in-part of Ser. No. 06/838,264, both entitled
"Provision of Density Specific Blood Cells for the Structuredness of the
Cytoplasmic Matrix (SCM) Test"; and (2) Ser. No. 06/867,079, filed May 27,
1986 (now abandoned), and Ser. No. 07/222,115, filed Jul. 20, 1988, a
continuation-in-part of Ser. No. 06/867,079, both entitled "Method for
Measuring Polarization of Bathochromically Shifted Fluorescence." The
disclosures of these related patent applications are incorporated herein
by this reference.
Claims
What is claimed is:
1. A substantially purified cancer recognition factor, the factor being a
peptide of nine amino acid residues to 35 amino acid residues including a
core sequence of nine amino acid residues having an amphipathicity profile
substantially equivalent to that of the sequence F-L-M-I-D-O-N-T-K,
wherein the sixth amino acid of the core sequence is selected from the
group consisting of Q and N, the seventh amino acid of the core sequence
is selected from the group consisting of N and Q, and the ninth amino acid
of the core sequence is selected from the group consisting of K and R, the
factor producing at least a ten percent (10%) decrease in the
intracellular fluorescence polarization value of lymphocytes capable of
responding in the structuredness of the cytoplasmic matrix (SCM) test as
isolated from donors afflicted with cancer.
2. The cancer recognition factor of claim 1 producing at least a 25 percent
decrease in the intracellular fluorescence polarization of SCM-responding
lymphocytes from donors afflicted with cancer.
3. The cancer recognition factor of claim 1 wherein the peptide comprises
from 9 to 35 amino acid residues, and wherein the core sequence of 9 amino
acid residues is F-X.sub.15 -M-X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20
-X.sub.21 -K, wherein X.sub.15 and X.sub.17 are each independently
selected from the group consisting of I, L, and V; X.sub.18 is selected
from the group consisting of D and E; X.sub.19 and X.sub.20 are each
independently selected from the group consisting of Q and N; and X.sub.21
is selected from the group consisting of S and T.
4. The cancer recognition factor of claim 1 wherein the core sequence of 9
amino acid residues is F-L-M-I-D-Q-N-T-K.
5. The cancer recognition factor of claim 1 wherein the peptide has the
sequence F-X -M-X.sub.17 -X.sub.18 - X.sub.19 - X.sub.20 -X.sub.21 -K,
wherein X.sub.15 and X.sub.17 are each independently selected from the
group consisting of I, L, and V; X.sub.18 is selected from the group
consisting of D and E; X.sub.19 and X.sub.20 are each independently
selected from the group consisting of Q and N; and X.sub.21 is selected
from the group consisting of S and T.
6. The cancer recognition factor of claim 5 wherein the peptide has the
sequence F-L-M-I-D-Q-N-T-K.
7. The cancer recognition factor of claim 1 wherein the peptide has the
sequence F-X.sub.9 -K-P-F-X.sub.13 -F-X.sub.15 -M-X.sub.17 -X.sub.18
-X.sub.19 -X.sub.20 -X.sub.21 -K, wherein X.sub.13, X.sub.15, and X.sub.17
are each independently selected from the group consisting of I, L, and V;
X.sub.18 is selected from the group consisting of D and E; X.sub.9,
X.sub.19, and X.sub.20 are each independently selected from the group
consisting of Q and N; and X.sub.21 is selected from the group consisting
of S and T.
8. The cancer recognition factor of claim 7 wherein the peptide has the
sequence F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K.
9. The cancer recognition factor of claim 1 wherein the peptide has the
sequence F-X.sub.9 -K-P-F-X.sub.13 -F-X.sub.15 -M-X.sub.17 -X.sub.18
-X.sub.19 -X.sub.20 -X.sub.21 -K-X.sub.23 -P-X.sub.25 -F-M-G-K, wherein
X.sub.13, X.sub.15, X.sub.17, X.sub.23, and X.sub.25 are each
independently selected from the group consisting of I, L and V; X.sub.18
is selected from the group consisting of D and E; X.sub.9, X.sub.19, and
X.sub.20 are each independently selected from the group consisting of Q
and N; and X.sub.21 is selected from the group consisting of S and T.
10. The cancer recognition factor of claim 9 wherein the peptide has the
sequence F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K.
11. The cancer recognition factor of claim 1 wherein the peptide has the
sequence M-X.sub.2 -P-P-X.sub.5 -X.sub.6 -K-F-X.sub.9 -K-P-F-X.sub.13
-F-X.sub.15 -M-X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20 -X.sub.21 -K,
wherein X.sub.2, X.sub.6, X.sub.13, X.sub.15, and X.sub.17 are each
independently selected from the group consisting of I, L, and V; X.sub.5
and X.sub.18 are each independently selected from the group consisting of
D and E; X.sub.9, X.sub.19, and X.sub.20 are each independently selected
from the group consisting of Q and N; and X.sub.21 is selected from the
group consisting of S and T.
12. The cancer recognition factor of claim 11 wherein the peptide has the
sequence M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K.
13. A substantially purified cancer recognition factor useful in the
structuredness of the cytoplasmic matrix (SCM) test consisting essentially
of a peptide have an amino acid sequence of M-X.sub.2 -P-P-X.sub.5
-X.sub.6 -K-F-X.sub.9 -K-P-F-X.sub.13 -F-X.sub.15 -M-X.sub.17 -X.sub.18
-X.sub.19 -X.sub.20 -X.sub.21 -K-X.sub.23 -P-X.sub.25 -F-M-G-K, wherein
X.sub.2, X.sub.6, X.sub.13, X.sub.15, X.sub.17, X.sub.23 and X.sub.25 are
each independently selected from the group consisting of I, L, and V;
X.sub.5 and X.sub.18 are each independently selected from the group
consisting of D and E; X.sub.9, X.sub.19, and X.sub.20 are each
independently selected from the group consisting of Q and N; and X.sub.21
is selected from the group consisting of S and T, the factor producing at
least a 10 percent decrease in the intracellular fluorescence polarization
value of SCM-responding lymphocytes from donors afflicted with cancer.
14. A substantially purified cancer recognition factor useful in the
structuredness of the cytoplasmic matrix (SCM) test consisting essentially
of a peptide of from 29 to 35 amino acid residues including a core
sequence at amino acids 14-22 of F-L-M-I-X.sub.18 -Q-N-T-K, wherein
X.sub.18 is selected from the group consisting of D and E, the factor
producing at least a 10 percent decrease in the intracellular fluorescence
polarization value of SCM-responding lymphocytes from donors afflicted
with cancer.
15. The cancer recognition factor of claim 14 wherein the peptide has the
sequence X.sub.1 -I-P-P-X.sub.5
-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-X.sub.23 -P-L-F-M-G-K, wherein X.sub.1
is selected from the group consisting of V, M, and S; X.sub.5 is selected
from the group consisting of E and D; and X.sub.23 is selected from the
group consisting of T and V.
16. The cancer recognition factor of claim 15 wherein the factor has the
sequence V-I-P-P-E-V=K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K.
17. The cancer recognition factor of claim 15 wherein the factor has the
sequence M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K.
18. The cancer recognition factor of claim 14 wherein the factor has the
sequence M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-P-C-F-M-G-C.
19. The cancer recognition factor of claim 14 wherein the factor has the
sequence X.sub.1 -I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-C-L-F-M-G-K,
wherein X.sub.1 is selected from the group consisting of M and V.
20. The cancer recognition factor of claim 19 wherein the factor has the
sequence M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-C-L-F-M-G-K.
21. The cancer recognition factor of claim 14 wherein the factor has the
sequence X.sub.1 -I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-R-P-L-F-M-G-K,
wherein X.sub.1 is selected from the group consisting of R and S.
22. The cancer recognition factor of claim 21 wherein the factor has the
sequence R-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-R-P-L-F-M-G-K.
23. The cancer recognition factor of claim 14 wherein the factor has the
sequence V-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-P-L-F-M-G-K.
24. The cancer recognition factor of claim 14 wherein the factor has the
sequence V-I-P-P-E-V-K-F-N-C-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K.
25. The cancer recognition factor of claim 14 wherein the peptide is
selected from the group consisting of peptides having amino acid sequences
X.sub.1 -I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-P-C-F-M-G-C and
X.sub.1
-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-P-C-F-M-G-C-V-V-N-C-T-E,
wherein X.sub.1 is selected from the group consisting of R and S.
26. The cancer recognition factor of claim 25 wherein the peptide has the
amino acid sequence
R-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-P-C-F-M-G-C.
27. The cancer recognition factor of claim 14 wherein the peptide is
selected from the group consisting of peptides having amino acid sequences
X.sub.1 -I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-E-Q-N-T-K-S-P-L-F-M-G-K and
X.sub.1
-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-E-Q-N-T-K-S-P-L-F-M-G-K-V-V-N-P-T-Q,
wherein X.sub.1 is selected from the group consisting of V and S.
28. The cancer recognition factor of claim 27 wherein the peptide has the
amino acid sequence
V-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-E-Q-N-T-K-S-P-L-F-M-G-K.
29. The cancer recognition factor of claim 14 wherein the factor has the
amino acid sequence X.sub.1
-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-E-Q-N-T-K-S-P-L-F-M-G-K-V-V-N-P-T-Q,
wherein X.sub.1 is selected from the group consisting of S and V.
30. The cancer recognition factor of claim 29 wherein the peptide has the
amino acid sequence
S-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-E-Q-N-T-K-S-P-L-F-M-G-K-V-V-N-P-T-Q.
31. A substantially purified cancer recognition factor active in the
structuredness of the cytoplasmic matrix (SCM) test consisting essentially
of low molecular weight peptide passing through filters with a nominal
1000-dalton molecular weight cutoff and retained by filters with a nominal
500-dalton molecular weight cutoff, the factor being substantially free of
intact .alpha..sub.1 -antitrypsin or other molecules larger than about
10,000 daltons, the factor producing at least a 10 percent decrease in the
intracellular fluorescence polarization value of SCM-responding
lymphocytes from donors afflicted with cancer as measured by the standard
SCM test.
32. The cancer recognition factor of claim 31 producing at least a 25
percent decrease in the intracellular fluorescence polarization value of
SCM-responding lymphocytes from donors afflicted with cancer as measured
by the standard SCM test.
33. A method for testing lymphocytes obtained from a mammalian donor for
the presence or absence of malignancy in the donor comprising the steps
of:
(a) contacting a suspension of the lymphocytes with the substantially
purified cancer recognition factor of claim 1, and
(b) determining the decrease in the structuredness of the cytoplasmic
matrix of the lymphocytes resulting from the step of contacting the
suspension of the lymphocytes.
34. The method of claim 33 wherein the step of determining the decrease in
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.C, for a second aliquot
of the lymphocytes that has not been contacted with the cancer recognition
factor; and
(c) determining the ratio of P.sub.S to P.sub.C, whereby a ratio of P.sub.S
to P.sub.C of less than about 0.9 indicates the presence of a malignancy
in the body of the donor of the lymphocytes.
35. The method of claim 33 wherein the step of determining the decrease in
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.M, for a second aliquot
of the lymphocytes that has been contacted with a mitogen selected from
the group consisting of phytohaemagglutinin, concanavalin A, and pokeweed
mitogen; and
(c) determining an SCM response ratio, R.sub.SCM, as the ratio of P.sub.S
to P.sub.M, whereby an RR.sub.SCM of less than about 0.9 indicates the
presence of a malignancy in the body of the donor of the lymphocytes.
36. A method for screening a blood sample for the presence of a malignancy
in the body of the donor of the blood sample comprising the steps of:
(a) separating potentially SCM-responding lymphocytes from the blood
sample;
(b) contacting the separated lymphocytes with the substantially purified
cancer recognition factor of claim 1 to stimulate the lymphocytes;
(c) contacting the stimulated lymphocytes with a fluorogenic agent
precursor for a sufficient time for the precursor to penetrate the
lymphocytes for intracellular enzymatic hydrolysis to a fluorogenic agent,
thereby generating stimulated fluorogenic agent-containing lymphocytes;
(d) exciting the stimulated fluorogenic agent-containing lymphocytes with
polarized light thereby causing the lymphocytes to fluoresce;
(e) measuring the vertically polarized and horizontally polarized
fluorescence emissions from the fluorescing lymphocytes for determining a
polarization value for the fluorescing lymphocytes; and
(f) comparing the determined polarization value for the stimulated
lymphocytes with a polarization value for a control aliquot of lymphocytes
from the same donor which has been subjected to the operation of steps
(a), (c), (d), and (e) but not (b), thereby to indicate the presence or
absence of cancer in the body of the donor of the lymphocytes.
37. The method of claim 36 wherein steps (b) and (c) occur simultaneously.
38. A method for testing lymphocytes obtained from a mammalian donor for
the presence or absence of malignancy in the donor comprising the steps
of:
(a) contacting a suspension of the lymphocytes with the substantially
purified cancer recognition factor of claim 13, and
(b) determining the decrease of the cytoplasmic matrix of the lymphocytes
resulting from the step of contacting the suspension of the lymphocytes.
39. The method of claim 38 wherein the step of determining the decrease in
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.C, for a second aliquot
of the lymphocytes that has not been contacted with the cancer recognition
factor; and
(c) determining the ratio of P.sub.S to P.sub.C, whereby a ratio of P.sub.S
to P.sub.C of less than about 0.9 indicates the presence of a malignancy
in the body of the donor of the lymphocytes.
40. The method of claim 38 wherein the step of determining the decrease in
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.M, for a second aliquot
of the lymphocytes that has been contacted with a mitogen selected from
the group consisting of phytohaemagglutinin, concanavalin A, and pokeweed
mitogen; and
(c) determining an SCM response ratio, R.sub.SCM, as the ratio of P.sub.S
to P.sub.M, whereby an RR.sub.SCM of less than about 0.9 indicates the
presence of a malignancy in the body of the donor of the lymphocytes.
41. A method for screening a blood sample for the presence of a malignancy
in the body of the donor of the blood sample comprising the steps of:
(a) separating potentially SCM-responding lymphocytes from the blood
sample;
(b) contacting the separated lymphocytes with the substantially purified
cancer recognition factor of claim 13 to stimulate the lymphocytes;
(c) contacting the stimulated lymphocytes with a fluorogenic agent
precursor for a sufficient time for the precursor to penetrate the
lymphocytes for intracellular enzymatic hydrolysis to a fluorogenic agent,
thereby generating stimulated fluorogenic agent-containing lymphocytes;
(d) exciting the stimulated fluorogenic agent-containing lymphocytes with
polarized light thereby causing the lymphocytes to fluoresce;
(e) measuring the vertically polarized and horizontally polarized
fluorescence emissions from the fluorescing lymphocytes for determining a
polarization value for the fluorescing lymphocytes; and
(f) comparing the determined polarization value for the stimulated
lymphocytes with a polarization value for a control aliquot of lymphocytes
from the same donor which has been subjected to the operation of steps
(a), (c), (d), and (e) but not (b), thereby to indicate the presence or
absence of cancer in the body of the donor of the lymphocytes.
42. The method of claim 41 wherein steps (b) and (c) occur simultaneously.
43. A method for testing lymphocytes obtained from a mammalian donor for
the presence or absence of malignancy in the donor comprising the steps
of:
(a) contacting a suspension of the lymphocytes with the substantially
purified cancer recognition factor of claim 14; and
(b) determining the decrease in the structuredness of the cytoplasmic
matrix of the lymphocytes resulting from the step of contacting the
suspension of the lymphocytes.
44. The method of claim 43 wherein the step of determining the decrease in
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.C, for a second aliquot
of the lymphocytes that has not been contacted with the cancer recognition
factor; and
(c) determining the ratio of P.sub.S to P.sub.C, whereby a ratio of P.sub.S
to P.sub.C of less than about 0.9 indicates the presence of a malignancy
in the body of the donor of the lymphocytes.
45. The method of claim 43 wherein the step of determining the decrease in
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.M, for a second aliquot
of the lymphocytes that has been contacted with a mitogen selected from
the group consisting of phytohaemagglutinin, concanavalin A, and pokeweed
mitogen; and
(c) determining an SCM response ratio, R.sub.SCM, as the ratio of P.sub.S
to P.sub.M, whereby an RR.sub.SCM of less than about 0.9 indicates the
presence of a malignancy in the body of the donor of the lymphocytes.
46. A method for screening a blood sample from the presence of a malignancy
in the body of the donor of the blood sample comprising the steps of:
(a) separating potentially SCM-responding lymphocytes from the blood
sample;
(b) contacting the separated lymphocytes with the substantially purified
cancer recognition factor of claim 14 to stimulate the lymphocytes;
(c) contacting the stimulated lymphocytes with a fluorogenic agent
precursor for a sufficient time for the precursor to penetrate the
lymphocytes for intracellular enzymatic hydrolysis to a fluorogenic agent,
thereby generating stimulated fluorogenic agent-containing lymphocytes;
(d) exciting the stimulated fluorogenic agent-containing lymphocytes with
polarized light thereby causing the lymphocytes to fluoresce;
(e) measuring the vertically polarized and horizontally polarized
fluorescence emissions from the fluorescing lymphocytes for determining a
polarization value for the fluorescing lymphocytes; and
(f) comparing the determined polarization value for the stimulated
lymphocytes with a polarization value for a control aliquot of lymphocytes
from the same donor which has been subjected to the operation of steps
(a), (c), (d), and (e) but not (b), thereby to indicate the presence or
absence of cancer in the body of the donor of the lymphocytes.
47. The method of claim 46 wherein steps (b) and (c) occur simultaneously.
48. A method for testing lymphocytes obtained from a mammalian donor for
the presence or absence of malignancy in the donor comprising the steps
of:
(a) contacting a suspension of the lymphocytes with the substantially
purified cancer recognition factor of claim 31; and
(b) determining the decrease in the structuredness of the cytoplasmic
matrix of the lymphocytes resulting from the step of contacting the
suspension of the lymphocytes.
49. The method of claim 48 wherein the steps of determining the decrease in
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.C, for a second aliquot
of the lymphocytes that has not been contacted with the cancer recognition
factor; and
(c) determining the ratio of P.sub.S to P.sub.C, whereby a ratio of P.sub.S
to P.sub.C of less than about 0.9 indicates the presence of a malignancy
in the body of the donor of the lymphocytes.
50. The method of claim 48 wherein the step of determining the decrease in
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.M, for a second aliquot
of the lymphocytes that has been contacted with a mitogen selected from
the group consisting of phytohaemagglutinin, concanavalin A, and pokeweed
mitogen; and
(c) determining an SCM response ratio, R.sub.SCM, as the ratio of P.sub.S
to P.sub.M, whereby an RR.sub.SCM of less than about 0.9 indicates the
presence of a malignancy in the body of the donor of the lymphocytes.
51. A method for screening a blood sample from the presence of a malignancy
in the body of the donor of the blood sample comprising the steps of:
(a) separating potentially SCM-responding lymphocytes from the blood
sample;
(b) contacting the separated lymphocytes with the substantially purified
cancer recognition factor of claim 14 to stimulate the lymphocytes;
(c) contacting the stimulated lymphocytes with a fluorogenic agent
precursor for a sufficient time for the precursor to penetrate the
lymphocytes for intracellular enzymatic hydrolysis to a fluorogenic agent,
thereby generating stimulated fluorogenic agent-containing lymphocytes;
(d) exciting the stimulated fluorogenic agent-containing lymphocytes with
polarized light thereby causing the lymphocytes to fluoresce;
(e) measuring the vertically polarized and horizontally polarized
fluorescence emissions from the fluorescing lymphocytes for determining a
polarization value for the fluorescing lymphocytes; and
(f) comparing the determined polarization value for the stimulated
lymphocytes with a polarization value for a control aliquot of lymphocytes
from the same donor which has been subjected to the operation of steps
(a), (c), (d), and (e) but not (b), thereby to indicate the presence or
absence of cancer in the body of the donor of the lymphocytes.
52. The method of claim 51 wherein steps (b) and (c) occur simultaneously.
53. A substantially purified cancer factor produced by a process comprising
the steps of:
(a) obtaining a body fluid from a donor afflicted with cancer;
(b) separating a first fraction of the body fluid comprising molecules
having an apparent molecular weight of greater than 1000 daltons as
determined by ultrafiltration through an ultrafilter with a nominal
molecular weight cutoff of 1000 daltons, from a second fraction comprising
molecules having an apparent molecular weight of less than 1000 daltons;
and
(c) purifying the second fraction to obtain a substantially pure cancer
recognition factor, wherein the step of purifying the second fraction
comprises the step of desalting the second fraction by loading the second
fraction on gel filtration column with a fractionation range of about 0 to
about 700 daltons and capable of separating the salts therefrom, eluting
the loaded material from the column with water, and collecting that
portion eluting at an elution volume between about 0.3 and about 0.5 times
the total chromatographic bed volume to increase the specific activity of
the cancer recognition factor in the collected desalted portion relative
to the specific activity in the second fraction.
54. The substantially purified cancer recognition factor of claim 53
wherein the process further comprises:
(d) loading the collected desalted portion on a gel filtration column
having a fractionation range up to about 1500 daltons to about 30,000
daltons;
(e) eluting the material loaded onto the column in step (d) from the column
with a weak aqueous solution of an ammonium salt; and
(f) collecting that portion eluting at an elution volume between about 0.4
times and about 0.6 times the total chronographic bed volume thereby
increasing the specific activity of the cancer recognition factor in the
collected eluate relative to the specific activity in the collected
desalted portion.
55. The substantially purified cancer recognition factor of claim 54,
wherein the process further comprises the steps of:
(g) loading the collected eluate from the gel filtration column on a
diethylaminoethyl cellulose anion-exchange column;
(h) eluting the material loaded onto the column in step (g) from the column
with an increasing concentration of an ammonium salt; and
(i) collecting that portion of the eluate eluting from the column at about
0.28 to about 0.31M of the ammonium salt, thereby increasing the specific
activity of the cancer recognition factor in the collected eluate from the
diethylaminoethyl cellulose anion-exchange column relative to the specific
activity in the eluate from the gel filtration column.
56. The substantially purified cancer recognition factor of claim 55,
wherein the process further comprises the step of purifying the cancer
recognition factor from the collected eluate from the diethylaminoethyl
cellulose anion-exchange column to substantial homogeneity by reverse
phase high pressure chromatography.
57. A method for testing lymphocytes obtained from a mammalian donor for
the presence or absence of malignancy in the donor comprising the steps
of:
(a) contacting a suspension of the lymphocytes with the substantially
purified cancer recognition factor of claim 6; and
(b) determining, by the structuredness of the cytoplasmic matrix (SCM)
test, the decrease in the structuredness of the cytoplasmic matrix of the
lymphocytes resulting from the step of contacting the suspension of the
lymphocytes, wherein a decrease of the structuredness of the cytoplasmic
matrix is indicative of the presence of malignancy.
58. The method of claim 57 wherein the step of determining the decrease in
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.C, for a second aliquot
of the lymphocytes that has not been contacted with the cancer recognition
factor; and
(c) determining the ratio of P.sub.S to P.sub.C, whereby a ratio of P.sub.S
to P.sub.C of less than about 0.9 indicates the presence of a malignancy
in the body of the donor of the lymphocytes.
59. The method of claim 57 wherein the step of determining the decrease in
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.M, for a second aliquot
of the lymphocytes that has been contacted with a mitogen selected from
the group consisting of phytohaemagglutinin, concanavalin A, and pokeweed
mitogen; and
(c) determining an SCM response ratio, R.sub.SCM, as the ratio of P.sub.S
to P.sub.M, whereby an RR.sub.SCM of less than about 0.9 indicates the
presence of a malignancy in the body of the donor of the lymphocytes.
60. A method for screening a blood sample from the presence of a malignancy
in the body of the donor of the blood sample comprising the steps of:
(a) separating potentially SCM-responding lymphocytes from the blood
sample;
(b) contacting the separated lymphocytes with the substantially purified
cancer recognition factor of claim 6 to stimulate the lymphocytes;
(c) contacting the stimulated lymphocytes with a fluorogenic agent
precursor for a sufficient time for the precursor to penetrate the
lymphocytes for intracellular enzymatic hydrolysis to a fluorogenic agent,
thereby generating stimulated fluorogenic agent-containing lymphocytes;
(d) exciting the stimulated fluorogenic agent-containing lymphocytes with
polarized light thereby causing the lymphocytes to fluoresce;
(e) measuring the vertically polarized and horizontally polarized
fluorescence emissions from the fluorescing lymphocytes for determining a
polarization value for the fluorescing lymphocytes; and
(f) comparing the determined polarization value for the stimulated
lymphocytes with a polarization value for a control aliquot of lymphocytes
from the same donor which has been subjected to the operation of steps
(a), (c), (d), and (e) but not (b), thereby to indicate the presence or
absence of cancer in the body of the donor of the lymphocytes.
61. The method of claim 60 wherein steps (b) and (c) occur simultaneously.
62. The cancer recognition factor of claim 1 wherein the peptide comprises
from 13 to 35 amino acid residues and wherein the core sequence comprises
13 amino acid residues, the core sequence being F-X.sub.13 -F-X.sub.15
-M-X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20 -X.sub.21 -K-X.sub.23 -P,
wherein X.sub.13 X.sub.15, X.sub.17, and X.sub.23 are each independently
selected from the group consisting of I, L and V; X.sub.18 is selected
from the group consisting of D and E; X.sub.19 and X.sub.20 are each
independently selected from the group consisting of Q and N; and X.sub.21
is selected from the group consisting of S and T.
63. The cancer recognition factor of claim 1 wherein the peptide comprises
from 18 to 35 amino acids, and wherein the core sequence comprises 18
amino acid residues, the core sequence being X.sub.9 -K-P-F-X.sub.13
-F-X.sub.15 -M-X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20 -X.sub.21
-K-X.sub.23 -P-X.sub.25 -F, wherein X.sub.13, X.sub.15, X.sub.17,
X.sub.23, and X.sub.25 are each independently selected from the group
consisting of I, L, and V; X.sub.18 is selected from the group consisting
of D and E; X.sub.19 and X.sub.20 are each independently selected from the
group consisting of Q and N; and X.sub.21 is selected from the group
consisting of S and T.
64. A method for testing lymphocytes obtained from a mammalian donor for
the presence or absence of malignancy in the donor comprising the steps
of:
(a) contacting a suspension of the lymphocytes with the substantially
purified cancer recognition factor of claim 3; and
(b) determining, by the structuredness of the cytoplasmic matrix (SCM)
test, the decrease in the structuredness of the cytoplasmic matrix of the
lymphocytes resulting from the step of contacting the suspension of the
lymphocytes, or wherein a decrease of the structuredness of the
cytoplasmic matrix is indicative of the presence of malignancy.
65. The method of claim 64 wherein the step of determining the decrease of
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.C, for a second aliquot
of the lymphocytes that has not been contacted with the cancer recognition
factor; and
(c) determining the ratio of P.sub.S to P.sub.C, whereby a ratio of P.sub.S
to P.sub.C of less than about 0.9 indicates the presence of a malignancy
in the body of the donor of the lymphocytes.
66. The method of claim 64 wherein the step of determining the decrease in
the structuredness of the cytoplasmic matrix comprises the steps of:
(a) measuring the fluorescence polarization, P.sub.S, for a first aliquot
of the lymphocytes that has been contacted with the cancer recognition
factor;
(b) measuring the fluorescence polarization, P.sub.M, for a second aliquot
of the lymphocytes that has been contacted with a mitogen selected from
the group consisting of phytohaemagglutinin, concanavalin A, and pokeweed
mitogen; and
(c) determining an SCM response ratio, R.sub.SCM, as the ratio of P.sub.S
to P.sub.M, whereby an RR.sub.SCM of less than about 0.9 indicates the
presence of a malignancy in the body of the donor of the lymphocytes.
67. A method for screening a blood sample from the presence of a malignancy
in the body of the donor of the blood sample comprising the steps of:
(a) separating potentially SCM-responding lymphocytes from the blood
sample;
(b) contacting the separated lymphocytes with the substantially purified
cancer recognition factor of claim 6 to stimulate the lymphocytes;
(c) contacting the stimulated lymphocytes with a fluorogenic agent
precursor for a sufficient time for the precursor to penetrate the
lymphocytes for intracellular enzymatic hydrolysis to a fluorogenic agent,
thereby generating stimulated fluorogenic agent-containing lymphocytes;
(d) exciting the stimulated fluorogenic agent-containing lymphocytes with
polarized light thereby causing the lymphocytes to fluoresce;
(e) measuring the vertically polarized and horizontally polarized
fluorescence emissions from the fluorescing lymphocytes for determining a
polarization value for the fluorescing lymphocytes; and
(f) comparing the determined polarization value for the stimulated
lymphocytes with a polarization value for a control aliquot of lymphocytes
from the same donor which has been subjected to the operation of steps
(a), (c), (d), and (e) but not (b), thereby to indicate the presence or
absence of cancer in the body of the donor of the lymphocytes.
68. The method of claim 67 wherein steps (b) and (c) occur simultaneously.
Description
TABLE OF CONTENTS
BACKGROUND
SUMMARY
DRAWINGS
DEFINITIONS
DESCRIPTION
I. ISOLATED AND PURIFIED GENERAL CANCER-ASSOCIATED SCM-RECOGNITION FACTORS
A. Purification
B. Structure of the Isolated Cancer-associated SCM-recognition Factor
II. SYNTHETIC CANCER-ASSOCIATED SCM-RECOGNITION FACTOR
A. Sequence of the Synthetic SCM Factor Molecule
B. Properties of the Synthetic SCM Factor
C. Production and Activity of Fragments of Synthetic SCM Factor III. USE OF
THE PURIFIED AND SYNTHETIC SCM FACTORS
A. Performance of the SCM Test
B. Immunochemical Uses of the SCM Factors
C. Detection of SCM-specific Receptors
D. DNA Sequences and Vectors
IV. RELATIONSHIP BETWEEN THE SCM FACTOR AND CANCER
A. Protection of Cancer Cells in Vivo by SCM Factor
B. Use of the SCM Factor in the Detection and Management of Cancer
EXAMPLES
Example 1--Initial Purification of the General Cancer-associated SCM Factor
from Blood Plasma
Example 2--SCM Activity of Initially Purified SCM Factor from Example 1
Example 3--Further Purification of the SCM Factor of Example 1
Example 4--SCM Activity of Further Purified Preparations of Example 3
Example 5--Final Purification of SCM Factor of Example 3 by RP-HPLC
Example 6--Alternative RP-HPLC Purification of SCM Factor
Example 7--SCM Activity of RP-HPLC Purified Preparations of Example 6
Example 8--Identification and Isolation of SCM-active Tryptic Peptides from
SCM Factor Purified from Blood Plasma of Patients with Breast Cancer and
Lung Cancer
Example 9--Use of the Isolated SCM Factor as the Challenging Agent in the
SCM Test
Example 10--Activity of the Tryptic Peptide of Example 8 in the SCM Test
Example 11--Cross-reactivity of Isolated SCM Factors
Example 12--Modification of the SCM Response by the Isolated SCM Factor
Example 13--Effect of the Isolated SCM Factor on Lymphocyte Cytotoxicity
Example 14--Amino Acid Sequences of Isolated SCM Factors
Example 15--SCM Activity of Synthetic SCM Factor
Example 16--Fragments of Synthetic SCM Factor
Example 17--Activity of Fragments of Synthetic SCM Factor of Example 16 in
the SCM Test
Example 18--Amphipathicity Profiles of SCM-active Peptides and Peptide
Fragments
Example 19--Induction of SCM Response Characteristic of Malignancy from
Healthy Donors by Synthetic SCM Factor
Example 20--Preparation of Antibodies to Synthetic SCM Factor
Example 21--ELISA Assay for SCM Factor
Example 22--Activity of Anti-SCM Antibodies
Example 23--Determination of SCM-factor Levels in Ultrafiltrates of Blood
Plasmas by ELISA Assay
Example 24--NH.sub.2 -terminal Amino Acid Sequences of SCM Factor Secreted
from Human Cancer Cells in Culture
Example 25--Reactivity of SCM Factor Secreted from Human Cancer Cells in
Culture with Anti-SCM-factor Antibodies
Example 26--Detection of SCM Factor in Human Cancer Cells in Culture by
ELISA Assay
Example 27--Effect of SCM Factor on DNA Synthesis
Example 28--Effect of SCM Factor on Inhibition of Serine Proteases by
.alpha.-1-PI Protease Inhibitor
Example 29--Interaction of Protease Inhibitor .alpha.-1-PI with SCM-factor
Receptors
Example 30--Inhibition of SCM Factor Synthesis
Example 31--Effect of Synthetic SCM Factor and Fragments Thereof on Natural
Cytotoxicity of Lymphocytes
BACKGROUND
Many diseases occurring in humans and animals can be detected by the
presence of foreign substances, particularly in the blood, the substances
being specifically associated with a disease or condition. Tests for
antigens or other such substances produced as a result of such diseases
show great promise as a diagnostic tool for the early detection of the
particular disease which produced the antigen or other substance.
Procedures for the detection of such substances must be reliable,
reproducible, and sensitive in order to constitute a practical diagnostic
procedure for health care providers. In addition, any such procedure
should be able to be carried out quickly and inexpensively by persons of
ordinary skill and training in laboratory procedures.
For example, in the treatment of the various malignancies that afflict
humans and animals, referred to generally as cancer, it is recognized that
early detection is the key to effective treatment, especially as most
therapeutic procedures are more effective and safer in relatively early
stages of cancer than in later stages. For example, many chemotherapeutic
drugs that are toxic to malignant cells are also toxic to normal cells,
and the higher doses required to cure or arrest more advanced cases of
cancer can cause uncomfortable and serious side effects. Also, surgery is
most often effective only before the disease has spread or metastasized.
Far too many cases of cancer are only discovered too late for effective
treatment.
Accordingly, there has been and continues to be a great need for reliable
tests that can diagnose cancer at early stages, and a great deal of
research effort has gone to this end. In this connection new tests and
procedures are being developed to effect early diagnosis of cancer.
One extremely desirable aspect of such a test is its ability either to
detect all types of cancer generally, or to detect specific types of
cancer, depending on the materials used. The former application of such a
test is very important in mass screenings of large patient populations, as
would be done in routine checkups. In such mass screenings a test
dependent on a particular type of cancer would not be desirable, as there
are literally hundreds, if not thousands, of types of cancer and a test
that could spot only one or a few types of the disease is far too likely
to miss many cases of cancer. In general, these patients would present
either no symptoms or vague generalized symptoms that could not be readily
linked to a particular type of cancer, so there would be no basis for
suspecting a particular type and administering a test specific for that
type.
In contrast, once the presence of malignancy is known or strongly
suspected, it would be desirable to have a test that could pinpoint the
particular type of malignancy present. Such a test could add greatly to
the efficiency of treatment, because many of the most effective cancer
therapies, such as chemotherapeutic agents, are only effective against one
type of cancer or at best, a narrow range of types, and the wrong
chemotherapy can do more harm than good.
In an effort to meet this need and to improve the diagnosis and early
detection of cancer in human and animal bodies, a test procedure has been
developed which involves the measurement of changes in the structuredness
of the cytoplasmic matrix (SCM) of living lymphocytes when exposed either
to phytohaemagglutinin or to cancer-associated antigens. This procedure
has been described in L. Cercek, B. Cercek, and C. I. V. Franklin,
"Biophysical Differentiation Between Lymphocytes from Healthy Donors,
Patients with Malignant Diseases and Other Disorders," Brit J. Cancer 29,
345-352 (1974), and L. Cercek and B. Cercek, "Application of the
Phenomenon of Changes in the Structuredness of Cytoplasmic Matrix (SCM) in
the Diagnosis of Malignant Disorders: a Review," Europ. J. Cancer 13,
903-915 (1977).
In accordance with this procedure, a subpopulation of potentially
SCM-responding lymphocytes is separated from a blood sample of the patient
being tested and the lymphocytes are incubated with malignant tissue or
extracts of malignant tissue. If the blood sample donor is afflicted with
a malignancy, there is a characteristic SCM response that can be
differentiated from the SCM response of lymphocytes from donors not
afflicted with a malignancy. The SCM response is determined by measuring
changes in intracellular fluorescein fluorescence polarization of the
SCM-responding lymphocytes.
The changes seen in the SCM test are believed to reflect changes in the
internal structure of the lymphocyte as the lymphocyte is activated for
synthesis. These changes are seen as a decrease in the fluorescence
polarization of the cells when polarized light is used to excite the
fluorescein present in the cells. Fluorescence polarization is a measure
of intracellular rigidity; the greater the intracellular mobility, the
less the measured fluorescence polarization. An observed decrease in
fluorescence polarization is thought to result mainly from changes in the
conformation of the mitochondria, the energy-producing organelles of the
cell. The change in the mitochondria is believed to result from the
contractions of the cristae or inner folds of the mitochondrial membrane.
The SCM reflects the forces of interaction between macromolecules and
small molecules such as water molecules, ions, adenosine triphosphate, and
cyclic adenosine phosphate. Perturbations of these interactions result in
changes in the SCM.
The SCM test is capable of responding to a relatively small quantity of
malignant cells. About 10.sup.9 cells in a person weighing 70 kg are
enough to cause the lymphocytes to respond in the SCM test in the
characteristic pattern of malignancy. In mice, when as few as
3.5.times.10.sup.5 Ehrlich ascites (tumor) cells are implanted, the
pattern of the response in the SCM test is altered; response to
cancer-specific antigens is induced, while the normal response to
phytohaemagglutinin is virtually eliminated (L. Cercek and B. Cercek,
"Changes in SCM-Responses of Lymphocytes in Mice After Implantation with
Ehrlich Ascites Cells," Europ. J. Cancer 17, 167-171 (1981)).
The SCM test allows early detection of cancer, often much earlier than is
possible by conventional methods, with relatively little discomfort to the
patient except as may be involved in taking a blood sample.
However, this procedure does have disadvantages. For example, it requires
preparation of crude extracts from tumor tissues and the like or the use
of the tumor tissue itself as a source of cancer-associated antigens.
There are several major problems with the use of malignant tissue or
extracts of such tissue in the SCM test. For example, it is sometimes
difficult to obtain the required quantity of tissue. Also, the use of
whole tissues or crude extracts of tissues can introduce interfering
substances into the test procedure. These interfering substances can
adversely affect the sensitivity of the test or adversely affect the test
results themselves. The presence or absence of these interfering
substances can easily vary from batch to batch of malignant tissue,
introducing undesirable variability into the SCM test. Additionally,
because the interfering substances are present in whole tissue or crude
extracts, they are very difficult to identify or quantitate.
Accordingly it is very desirable to identify, separate, and purify the
factor or factors that provoke a response by SCM-responding lymphocytes.
The use of such purified factor or factors would enhance the SCM cancer
screening test because interfering substances would not be present, and
would aid in the study of cancer, its causes and its effects on human and
animal bodies. The availability of purified factors would allow the
production of specific antibodies against them. Such antibodies would be
useful for both diagnosis and treatment of cancer.
It is also very desirable to determine the complete chemical composition
and structure of such SCM-active factors. If they turn out to be peptides
or proteins, it would be especially desirable to determine their complete
amino acid sequence. The knowledge of their complete amino acid sequence
would allow their production by either solid-phase peptide synthesis
techniques or recombinant DNA techniques. The application of these
techniques would result in the availability of larger quantities of the
factors without the necessity of isolating them from blood plasma or
cancer tissue.
SUMMARY
We have discovered cancer recognition factors in body fluids, in particular
in blood plasma, and purified these factors to substantial homogeneity.
These factors produce a response in SCM-responding lymphocytes obtained
from a donor with cancer that is identical to the response produced in
such lymphocytes by cancer-associated extracts and/or tumor tissue in the
SCM test. As described herein, the factors are designated in the singular
and are referred to herein as the "cancer recognition factor useful in the
structuredness of the cytoplasmic matrix (SCM) test," as the "cancer
recognition factor," or merely as the "SCM factor."
The activity of the SCM factor can be demonstrated at a number of stages of
purification of the factor from plasma, beginning with a step of
ultrafiltration. In this step molecules with an apparent molecular weight
of less than 1,000 daltons are separated from molecules with a larger
molecular weight by ultra filtration through a filter with a nominal
molecular weight cutoff of 1,000 daltons. The SCM factor is found in the
fraction passing through the filter, in contrast to most other peptides
and all proteins. The factor consists essentially of low molecular weight
peptide passing through such filters and producing at least a ten percent
decrease in the intracellular fluorescence polarization value of
SCM-responding lymphocytes isolated from donors afflicted with cancer when
used to challenge lymphocytes in the standard SCM test.
Further purification of the SCM factor, as described below, results in a
substantially homogeneous peptide of 29 to 35 amino acid residues. Because
the SCM factors isolated from blood plasma samples obtained from patients
with different types of cancer were largely homologous, a synthetic
29-amino-acid peptide, designated "synthetic SCM factor," was prepared.
This peptide was fully active in the SCM test; certain fragments of this
peptide, as described below, were also active in the SCM test.
1. Peptides Possessing SCM-Factor Activity
As determined from studies on fragments of synthetic SCM factor, a peptide
of at least 9 amino acid residues including a core sequence of 9 amino
acid residues having an amphipathicity profile substantially equivalent to
that of the sequence F-L-M-I-D-Q-N-T-K is expected to have SCM-factor
activity and to produce at least a 10 percent decrease in the
intracellular fluorescence polarization value of SCM-responding
lymphocytes from donors afflicted with cancer.
The core sequence of 9 amino acid residues can be F-X.sub.15 -M-X.sub.17
-X.sub.18 -X.sub.19 -X.sub.20 -X.sub.21 -K. In this sequence, X.sub.15 and
X.sub.17 are each independently selected from the group consisting of I,
L, and V; X.sub.18 is selected from the group consisting of D and E;
X.sub.19 and X.sub.20 are each independently selected from the group
consisting of Q and N; and X.sub.21 is selected from the group consisting
of S and T. These substitutions are examples of "conservative" amino acids
substitutions, in which substitution of one of the amino acids of the
group for another amino acid is expected to cause essentially no change in
the structure of activity of the peptide because the properties of the
amino acids are so similar. In particular, the core sequence can be
F-L-M-I-D-Q-N-T-K.
Determination of the amino acid sequences of purified SCM factor obtained
from blood plasma with patients with different types of cancer has led to
the conclusion that such factors consist essentially of a peptide of from
29 to 35 amino acid residues including a core sequence at amino acid
residues 14-22 of F-L-M-I-X.sub.18 -Q-N-T-K, where X.sub.18 is D or E.
Particular examples of peptides conforming to this general sequence pattern
and having SCM-factor activity include:
(1) X.sub.1 -I-P-P-X.sub.5 -V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-X.sub.23
-P-L-F-M-G-K, where: X.sub.1 can be V, M, or S; X.sub.5 can be E or D, and
X.sub.23 can be T or V; particular peptides of this sequence pattern are
V-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K and
M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K;
(2) M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-P-C-F-M-G-C;
(3) X1-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-C-L-F-M-G-K, where
X.sub.1 can be M or V, typically M;
(4) X -I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-R-P-L-F-M-G-K, where
X.sub.1 can be R or S, typically S;
(5) V-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-P-L-F-M-G-K;
(6) V-I-P-P-E-V-K-F-N-C-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K;
(7) X.sub.1 -I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-P-C-F-M-G-C or X
-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-C-P-C-F-M-G-C-V-V-N-C-T-E,
where X.sub.1 is R or S, where the sequence typically has 29 amino acids
and X.sub.1 is typically R;
(8) X.sub.1 -I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-E-Q-N-T-K-S-P-L-F-M-G-K or
X.sub.1
-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-E-Q-N-T-K-S-P-L-F-M-G-K-V-V-N-P-T-Q,
where X.sub.1 is V or S, where the sequence typically has 29 amino acids
and X.sub.1 is typically V; and
(9) X1-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-E-Q-N-T-K-S-P-L-F-M-G-K-V-V-N-P-T-Q,
where X.sub.1 is S or V, typically S.
These sequences have considerable homology. Therefore, a "consensus"
sequence of 29 amino acids,
M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K, has been
synthesized. This "consensus" sequence is identical with one of the
sequences determined from sequence analysis of preparations of SCM factors
purified from blood plasma of a cancer patient, specifically, from
patients with colon and lung cancer. Because substitution of certain amino
acids for other amino acids in this sequence, as discussed above, is not
expected to substantially alter the activity of the SCM factor, the
following sequence derived from the "consensus" sequence by conservative
amino acid substitutions is also expected to have SCM-factor activity:
M-X.sub.2 -P-P-X.sub.5 -X.sub.6 -K-F-X.sub.9 -K-P-F-X.sub.13 -F-X.sub.15
-M-X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20 -X.sub.21 -K-X.sub.23
-P-X.sub.25 -F-M-G-K, in which X.sub.2, X.sub.6, X.sub.13, X.sub.15,
X.sub.17, X.sub.23, and X.sub.25 are each independently selected from the
group consisting of I, L, and V; X.sub.5 and X.sub.18 are each
independently selected from the group consisting of D and E; X.sub.9,
X.sub.19 and X.sub.20 are each independently selected from the group
consisting of Q and N; and X.sub.21 is selected from the group consisting
of S and T.
Additionally, particular fragments of the consensus sequence or of peptides
derived from the consensus sequence by conservative amino acid
substitution are known (in the case of fragments of the consensus sequence
itself) or expected (in the case of fragments of peptides derived from the
consensus sequence by conservative amino acid substitution) to have
SCM-factor activity. The sequences that represent fragments of the
consensus sequence are, respectively: F-L-M-I-D-Q-N-T-K (amino acid
residues 14-22)-; F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K (amino acid residues
8-22); F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K (amino acid residues
8-29); and M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K (amino acid
residues 1-22). The shortest of these peptides, F-L-M-I-D-Q-N-T-K,
represents the core sequence itself.
The following peptides, derived from these peptides by conservative amino
acid substitution, are also expected to have SCM-factor activity:
(1) F-X.sub.15 -M-X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20 -X.sub.21 -K,
wherein X.sub.15 and X.sub.17 are each independently selected from the
group consisting of I, L, and V; X.sub.18 is selected from the group
consisting of D and E; X.sub.19 and X.sub.20 are each independently
selected from the group consisting of Q and N; and X.sub.21 is selected
from the group consisting of S and T;
(2) F-X.sub.9 -K-P-F-X.sub.13 -F-X.sub.15 -M-X.sub.17 -X.sub.18 -X.sub.19
-X.sub.20 -X.sub.21 -K, wherein X.sub.13, X.sub.15 and X.sub.17 are each
independently selected from the group consisting of I, L, and V; X.sub.18
is selected from the group consisting of D and E; X.sub.9, X.sub.19 and
X.sub.20 are each independently selected from the group consisting of Q
and N; and X.sub.21 is selected from the group consisting of S and T;
(3) F-X.sub.9 -K-P-F-X.sub.13 -F-X.sub.15 -M-X.sub.17 -X.sub.19 -X.sub.20
-X.sub.21 -K-X.sub.23 -P-X.sub.25 -F-M-G-K, wherein X.sub.13, X.sub.15,
X.sub.17, X.sub.23, and X.sub.25 are each independently selected from the
group consisting of I, L, and V; X.sub.18 is selected from the group
consisting of D and E; X.sub.9, X.sub.19 and X.sub.20 are each
independently selected from the group consisting of Q and N; and X.sub.21
is selected from the group consisting of S and T; and
(4) M-X.sub.2 -P-P-X.sub.5 -X.sub.6 -K-F-X.sub.9 -K-P-F-X.sub.13
-F-X.sub.15 -M-X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20 -X.sub.21 -K,
wherein X.sub.2, X.sub.6, X.sub.13, X.sub.15 and X.sub.17 are each
independently selected from the group consisting of I, L, and V; X.sub.5
and X.sub.18 are each independently selected from the group consisting of
D and E; X.sub.9, X.sub.19 and X.sub.20 are each independently selected
from the group consisting of Q and N; and X.sub.21 is selected from the
group consisting of S and T.
2. Purification of Peptides Possessing SCM-Factor Activity
The SCM factor can be purified by ultrafiltering a body fluid from a donor
afflicted with cancer in order to separate a first fraction of the body
fluid comprising molecules having an apparent molecular weight greater
than 1,000 daltons from a second fraction comprising molecules having an
apparent molecular weight of less than 1,000 daltons. The body fluid is
selected from the group consisting of peripheral blood, urine, and plasma.
Preferably, after ultrafiltration the factor undergoes a further
purification process comprising several stages, each stage resulting in a
more highly purified factor.
The first stage of this purification process comprises elution from a gel
filtration column with a fractionation range of from 0 to about 700
daltons and capable of separating the salts from the ultrafiltrate, the
factor eluting at between about 0.3 and about 0.5 times the total
chromatographic bed volume.
The second stage comprises elution from a gel filtration column having a
fractionation range of from about 1500 daltons to about 30,000 daltons,
the factor eluting from such a column at between about 0.4 and about 0.6
times the total chromatographic bed volume.
The next stage of this purification process comprises elution from an
anion-exchange column of diethylaminoethyl cellulose at between about
0.28M to about 0.31M of ammonium bicarbonate.
The final stage comprises purifying the factor to substantial homogeneity
by reverse-phase high-pressure liquid chromatography.
Although it is preferred to use the more highly purified preparations of
the factor in the SCM test, the factor from any stage of the purification,
including the initial ultrafiltrate, can be used in the test.
3. DNA Sequences Coding for the SCM Factor
DNA sequences encoding the SCM factor as described above are useful for
both diagnostic purposes and for production of large quantities of SCM
factor by recombinant DNA procedures.
Generally, the desired DNA sequence encodes the SCM factor in isolation
from DNA encoding proteins normally accompanying SCM factor. Particularly
important are DNA sequences encoding the core sequence of
F-L-M-I-D-Q-N-T-K and the consensus sequence of
M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K.
The DNA sequence can be operably linked to control sequences effective in
expressing the DNA encoding the SCM factor in compatible host cells. This
DNA sequence, incorporating the control sequence, can be included in a
vector capable of transfecting at least some of the host cells within
which the DNA can be expressed. Host cells transfected with this vector
are also within the scope of the invention.
4. Antibodies Specific for the SCM Factor and Their Use
Antibodies specific for the substantially purified SCM factors can be
prepared by standard methods of preparing antibodies to peptides and are
within the scope of the invention. Particularly important are antibodies
specific for the core sequence and for the consensus sequence. The
antibody can be a monoclonal antibody prepared by methods known in the
art.
These antibodies can be used in a method for determining the level of SCM
factor in a body fluid. The method comprises:
(1) mixing the body fluid and the antibody; and
(2) determining the extent of reaction between the SCM factor in the body
fluid and the antibody by performing an immunoassay. The immunoassay can
be a radioimmunoassay, a fluorescence immunoassay, a chemiluminescence
immunoassay, an enzyme-linked immunoassay, or an immunoassay dependent on
aggregation of antigen-antibody complexes.
5. Use of Labeled SCM to Detect Specific Receptors
As an alternative to the SCM test for the detection of cancer, labeled SCM
factor can be used to determine the presence of receptors specific for SCM
factor on SCM-responding lymphocytes and the sensitization of the
lymphocytes to the SCM factor. The method for determining the presence of
receptors comprises:
(1) isolating SCM-responding lymphocytes;
(2) washing the isolated SCM-responding lymphocytes;
(3) incubating the isolated SCM-responding lymphocytes with a saturating
quantity of a natural or synthetic SCM factor labeled with a detectable
label; and
(4) determining the extent of the binding of the labeled SCM factor to the
SCM-responding lymphocytes in order to determine the presence of receptors
specific for SCM factor on the lymphocytes and the sensitization of the
lymphocytes to SCM factor.
6. Use of the SCM Factor in the SCM Test
The SCM factor according to the present invention can be used to detect
cancer in the SCM test. When using an SCM factor purified from a body
fluid of a patient with cancer, it is preferred to use a more highly
purified preparation of the factor, particularly a preparation of the
factor purified to substantial homogeneity by high-pressure liquid
chromatography. However, the factor from any stage of the purification,
including the initial ultrafiltrate, can be used in the test.
The SCM factor produces a positive response in the SCM test in a limited
fraction of lymphocytes, known as "SCM-responding lymphocytes," derived
from donors having a variety of different types of malignancies,
regardless of the type of cancer present in the donor from which the SCM
factor is isolated; the factor produces essentially no response in the SCM
test in lymphocytes derived from donors free of malignancy. This feature
makes the SCM factor useful for the general screening of blood samples or
other body fluid samples for the presence of malignancy.
Most generally, the method for testing lymphocytes from a mammalian donor
for the presence or absence of malignancy in the donor comprises:
(1) contacting a suspension of the lymphocytes with a substantially
purified cancer recognition factor (SCM factor) as described above; and
(2) determining the decrease in the structuredness of the cytoplasmic
matrix of the lymphocytes resulting from the step of contacting the
suspension of the lymphocytes.
A preferred method for quantifying the decrease in structuredness comprises
the steps of: (1) measuring the fluorescence polarization, P.sub.S, for an
aliquot of the lymphocytes that has been contacted with the cancer
recognition factor; (2) measuring the fluorescence polarization, P.sub.C,
for a control aliquot of the lymphocytes that has not been contacted; and
(3) determining the ratio of P.sub.S to P.sub.C. A ratio of P.sub.S to
P.sub.C lower than about 0.9 indicates a positive response to the SCM
factor and the presence of a malignancy in the donor of the lymphocytes.
A more preferred method comprises comparing P.sub.S to the fluorescence
polarization of another aliquot of the lymphocytes contacted with a
mitogen, P.sub.M, typically phytohaemagglutinin, to determine an SCM
response ratio, RR.sub.SCM, where:
RR.sub.SCM =P.sub.S /P.sub.M.
An RR.sub.SCM of less than about 0.9 indicates the presence of a malignancy
in the donor of the lymphocytes.
Alternatively, the method of employing the SCM factor in the SCM test can
comprise the steps of: (1) separating potentially SCM-responding
lymphocytes from the blood sample; (2) contacting the separated
lymphocytes with a cancer recognition factor to stimulate the lymphocytes;
(3) contacting the stimulated lymphocytes with a fluorogenic agent
precursor, defined as a nonfluorogenic compound hydrolyzable
intracellularly to a fluorogenic agent, to penetrate the lymphocytes for
intracellular enzymatic hydrolysis to the fluorogenic agent, thereby
generating stimulated fluorogenic agent-containing lymphocytes; (4)
exciting the stimulated fluorogenic agent-containing lymphocytes with
polarized light thereby causing them to fluoresce; (5) measuring the
vertically and horizontally polarized fluorescence emissions from the
fluorescing lymphocytes for determining a polarization value for the
fluorescing lymphocytes; and (6) comparing the determined polarization
value for the stimulated fluor-containing lymphocytes with the
polarization value for a control aliquot of lymphocytes from the same
donor, thereby to indicate the presence or absence of cancer in the body
of the donor of the lymphocytes. Steps (3) and (4) can occur
simultaneously.
The lymphocytes can be excited with vertically polarized light. The
polarization values when vertically polarized light is used are determined
in a fluorescence spectrophotometer in accordance with the relationship:
##EQU1##
where, I.sub.V and I.sub.H are the polarized fluorescence intensities in
the vertical and horizontal planes, respectively; and G is a correction
factor for the unequal transmission of the horizontal and vertical
components of the polarized light through the optical system of the
spectrophotometer.
7. Use of the SCM Factor in the Treatment of Cancer
Not only does the present invention provide a diagnostic technique for
identifying subjects afflicted with cancer, it also comprehends methods
for the treatment of such subjects. These methods are based on several
observations described in detail below. These observations reveal that the
SCM factor is produced by cancer cells, and that it has several effects:
enhancement of DNA synthesis, protection of cancer-associated proteases
against inhibition by their natural inhibitor .alpha.-1-PI and suppression
of the natural cytotoxicity of killer lymphocytes against malignant cells.
Such suppression of the natural cytotoxicity of killer lymphocytes can be
the result of SCM factor action at various steps of the immune defense
mechanism. Such SCM factor action can include decreasing the ability of
the effector killer lymphocytes to form a complex with the target cancer
cells or blocking of the signal transducing mitogen receptors in
lymphocytes, thus decreasing production of cytolysins such as tumor
necrosis factor (TNF) and other cytotoxic and/or cytolytic molecules. SCM
factor action can also include direct interaction with and inactivation of
such cytolytic molecules, and the scavenging of peroxides and other
oxygen-containing reactive species produced by various leukocytes against
cancer cells.
Because the SCM factor appears to protect cancer cells in several ways,
reduction of the in vivo activity of the SCM factor should increase the
efficiency of immunological surveillance by lymphocytes against malignant
cells.
Most generally, this treatment method is a method of treating a cancer
patient where at least one of the body fluids of the patient contains a
cancer recognition factor. The factor is a peptide of at least 9 amino
acid residues including a core sequence of 9 amino acid residues having an
amphipathicity profile substantially equivalent to that of the sequence
F-L-M-I-D-Q-N-T-K. The method comprises:
(1) treating a body fluid containing the cancer recognition factor to
reduce the in vivo effect of the factor by selectively inactivating it;
and
(2) returning the body fluid to the patient, thereby to enhance the
resistance of the patient to the cancer.
The body fluid can be peripheral blood. In this case, the step of treating
the body fluid can comprise dialysis of the peripheral blood to remove
peptides with an apparent molecular weight of less than 1,000 daltons.
This selectively inactivates the cancer recognition factor by its removal
from the blood.
Alternatively, the step of treating the body fluid can comprise
neutralizing the cancer recognition factor in the body fluid with an
antibody specific for it, or with univalent antigen-binding fragments of
the antibody, such as Fab fragments or Fab fragments.
As another alternative, the step of treating the body fluid can comprise
inactivating the cancer recognition factor with an antisense peptide whose
amino acid sequence is that encoded by the antisense strand of a DNA
sequence whose sense strand encodes an SCM factor.
These methods can further comprise the step of treating the body fluid with
a natural or synthetic protease inhibitor non-homologous with .alpha.-1-PI
protease inhibitor and non-homologous with any other protease inhibitor
that is substantially inhibited by SCM factor, the protease inhibitor used
for treatment being capable of inhibiting cancer-associated proteases
protected against .alpha.-1-PI inhibition by SCM factor. They can also
further comprise the step of treating the patient with a clinically
acceptable metabolic inhibitor, such as ascorbic acid, that causes a
decrease in production of the SCM factor by tumor cells.
Because the SCM factor is produced by cancer cells and is found in them, an
alternative method of treatment involves directing an anti-cancer
substance to cancer cells. This can comprise:
(1) tagging an antibody specific for SCM factor with the anti-cancer
substance; and
(2) administering the tagged antibody to a cancer patient so that the
tagged antibody can bind to cancer cells of the patient, thereby directing
the anti-cancer substance to the cancer cells. The antibody can be a
monoclonal antibody.
Another alternative method of treatment focuses on the reversal of the
NK-suppressive effect caused by SCM factor. As detailed below, this
NK-suppressive effect can be localized to a particular region of the SCM
factor--the carboxyl-terminal 22 residues. Accordingly, a method of
reversing the NK-suppressive action of SCM factor in vivo can comprise
administering to a patient at least one of whose body fluids contains SCM
factor a SCM-factor-inhibiting substance in a quantity sufficient to
substantially reverse the NK-suppressive action of the SCM factor and
substantially restore normal NK activity of lymphocytes of the patient as
measured by in vitro lysis of K562 cells by the lymphocytes. The
SCM-factor-inhibiting substance can be antibodies to SCM factor, univalent
antigen-binding fragments of antibodies to SCM factor, or antisense
peptides whose amino acid sequences are those encoded by the antisense
strand of DNA sequences whose sense strand encodes a NK-suppressive
sequence as described below under "Use of the SCM Factor in Suppressing
Natural Killer Activity."
8. Use of the SCM Factor in Imaging Cancer Cells
Because SCM factor is found in cancer cells, antibodies to SCM factor can
also be used to image cancer cells, particularly for diagnostic purposes.
The method comprises:
(1) labeling the antibody with an imaging substance; and
(2) utilizing the labeled antibody to image cancer cells by exposing the
cancer cells to the labeled antibody.
9. Use of the SCM Factor in Suppressing Natural Killer Activity
Both the substantially purified SCM factor and the synthetic SCM factor
suppress the natural killer (NK) activity of lymphocytes. This
NK-suppressive activity was found to be localized in amino acid residues
8-29 of the synthetic SCM factor, with an amino acid sequence of
F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K. Therefore, in accordance with
the conservative amino acid substitutions described above, a substantially
purified peptide of at least 22 amino acid residues including a natural
killer-suppressive (NK-suppressive) sequence of F-X.sub.9 -K-P-F-X.sub.13
-F-X.sub.15 -M-X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20 -X.sub.21
-K-X.sub.23 -P-X.sub.25 -F-M-G-K, wherein X.sub.13, X.sub.15, X.sub.17,
X.sub.23, and X.sub.25 are each independently selected from the group
consisting of I, L, and V; X.sub.18 is selected from the group consisting
of D and E; X.sub.9, X.sub.19 and X.sub.20 are each independently selected
from the group consisting of Q and N; and X.sub.21 is selected from the
group consisting of S and T is expected to have NK-suppressive activity.
Other fragments of the synthetic SCM factor, including fragment F1
(residues 1-22), F3 (residues 8-22), F4 (residues 14-22), F5 (residues
1-13), F7 (residues 14-29), and F8 (residues 23-29) had no NK-suppressive
activity.
The ability of SCM factor or portions of SCM factor to exert NK-suppressive
activity makes possible a method for assessing the effectiveness of an
anti-cancer agent capable of inhibiting the growth of malignant cells on a
cell culture. In this method, the cell culture includes both lymphocytes
exhibiting NK activity and malignant cells. The method comprises:
(1) incubating the cell culture with the substantially purified
NK-suppressive peptide in a quantity sufficient to substantially suppress
the NK activity of the lymphocytes of the cell culture;
(2) adding the anti-cancer agent to the cell culture in a quantity
sufficient to inhibit the growth of the malignant cells; and
(3) determining the effect of the anti-cancer agent on the malignant cells
by observing the inhibition of growth of the malignant cells caused by the
anti-cancer agent in the essential absence of NK activity caused by the
lymphocytes.
The NK-suppressive peptide or the entire SCM factor molecule can be used to
modulate the activity of the immune system. Such modulation can be
desirable in preventing rejection of transplants. The substantially
purified NK-suppressive peptide can be used to suppress the NK activity of
lymphocytes by administering it to the lymphocytes in a quantity
sufficient to substantially suppress the NK activity of the lymphocytes as
measured by the in vitro lysis of K562 cells. Similarly, a method for
inducing immunosuppression in vivo can comprise administering an
immunosuppressive fraction alone or in combination with a pharmaceutically
acceptable carrier in a quantity sufficient to create a degree of
immunosuppression capable of enhancing allograft survival. The
immunosuppressive fraction can be a substantially purified natural or
synthetic SCM factor or a NK-suppressive peptide.
DRAWINGS
These and other features, aspects, and advantages of the present invention
will become better understood with reference to the following description,
appended claims, and the accompanying drawings where:
FIG. 1 shows the amphipathicity profile of the SCM-active F4 fragment of
the synthetic SCM factor, representing amino acid residues 14-22 of the
synthetic SCM factor;
FIG. 2 shows the amphipathicity profile of the SCM-active octapeptide whose
sequence is F-W-G-A-E-G-O-R and which has been previously found to occur
as an impurity in some preparations of experimental allergic
encephalitogenic peptide (EAE peptide);
FIG. 3 is a schematic depiction of one form of ELISA assay for the SCM
factor;
FIG. 4 shows the results obtained from an experiment in which the
reactivity of antiserum raised against unconjugated SCM factor, as
determined by absorbance at 405 nm in a version of the ELISA assay, was
measured as a function of the dilution of the antiserum; and
FIG. 5 shows the results obtained from an experiment in which the
reactivity of antiserum raised against SCM factor conjugated with keyhole
limpet hemocyanin (KLH), as determined by absorbance at 405 nm in a
version of the ELISA assay, was measured as a function of the dilution of
the antiserum.
DEFINITIONS
Definitions for a number of terms which are used in the following
Description, Examples, and appended claims are collected here for
convenience.
To the extent that these definitions may vary from meanings within the art,
the definitions below are to control.
"General": Nonspecific with respect to the particular type of cancer
afflicting either the donor of the body fluid from which the SCM factor of
the present invention is purified, or the donor of the lymphocytes used
with that factor in the SCM test.
"Fluorogenic Agent Precursor": A nonfluorogenic compound capable of being
taken up by lymphocytes and converted intracellularly by hydrolysis into a
fluorogenic compound, of which the example used herein is fluorescein
diacetate (FDA).
"Standard SCM Test": An SCM test using 1.0 ml of a lymphocyte suspension at
6.times.10.sup.6 cells/ml and 0.1 ml of the cancer recognition factor or
mitogen, with FDA as the fluorogenic agent precursor and using an
excitation wavelength of 470 nm and an emission wavelength of 510 nm for
fluorescence polarization measurements.
"Apparent Molecular Weight" and "Nominal Molecular Weight Cutoff": Both of
these terms refer to the fact that the separation of molecules by
ultrafiltration according to size is approximate for molecules in the size
range of SCM factor, and depends on conformation as well as size. Thus an
ultrafilter with a nominal molecular weight cutoff of x daltons will
separate molecules with an apparent molecular weight of less than x
daltons from molecules with an apparent molecular weight greater than x
daltons. However, some molecules with an actual molecular weight greater
than x daltons will pass through such a filter.
"Substantially Pure Cancer Recognition Factor": Material exhibiting cancer
recognition activity as determined in the SCM test and of such a state of
purity that at least about 95% of other molecules with specific biological
activity, including all proteins and larger peptides, is not present in
the material. The term "substantially purified" refers to the same state
of purity.
"Tryptic Peptide": A peptide cleaved from a larger peptide by the action of
the proteolytic enzyme trypsin, which breaks peptide chains after lysine
or arginine residues.
DESCRIPTION
This invention relates to our discovery and purification to substantial
homogeneity of twelve peptides that are general cancer-associated
SCM-recognition factors having sera isolated from a number of patients
from different types of cancer. These peptides are all between 29 and 35
amino acids in length, crossreact in the SCM test, and show a striking
homology in amino acid sequence. This homology is so striking that a
29-amino acid peptide representing a consensus sequence of the twelve
purified peptides has been synthesized. This peptide, designated as
"synthetic SCM factor", has the amino acid sequence
M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K. This synthetic
peptide shares all of the properties of the general cancer-associated
SCM-recognition factor isolated from lymphocytes, including activity in
the SCM test and immunochemical reactivity. Even more unexpectedly, a
region of nine amino acids within that sequence, amino acids 14-22, with
the sequence F-L-M-I-D-Q-N T-K, is equally active in the SCM test. Other
partial sequences, including amino acids 8-22, 8-29, and 1-22, that
incorporate the 14-22 sequence are also fully active.
Biological properties of both the natural purified and the synthetic SCM
factors are described. These properties are substantially identical, as
far as has been determined, for the natural and synthetic SCM factors, and
include: (1) the ability of the SCM factor to modify the SCM responses of
lymphocytes from donors free of malignancy; (2) cross-reactivity of the
factor isolated from donors with various types of cancer in the SCM test;
(3) its ability to suppress the in vitro natural cytotoxicity of killer
lymphocytes toward malignant cells; and (4) the newly discovered property
of the SCM factor of protecting proteases that are believed to aid in the
proliferation and invasion of cancer cells from inhibition by the natural
inhibitor of those proteases, .alpha.-1-PI. Furthermore, the homology of
the SCM factor with .alpha.-1-PI has been discovered, along with the fact
that .alpha.-1-PI can reverse the responses in vitro of SCM-responding
lymphocytes of cancer patients to the SCM factor. A method for
purification of the SCM factor from blood plasma to substantial
homogeneity is described, as are methods of using both the synthetic SCM
factor and the SCM factor purified from plasma as challenging agents in
the SCM test. Lymphocyte receptor assays and various immunochemical
assays, including ELISA assays, are also described, as are DNA sequences
coding for the SCM factors and vectors incorporating these sequences.
Finally, methods of using the SCM factors in the management of cancer are
described.
I. ISOLATED AND PURIFIED GENERAL CANCER-ASSOCIATED SCM-RECOGNITION FACTORS
The general cancer-associated SCM-recognition factor was isolated and
purified to homogeneity from blood plasma obtained from patients with
twelve different types of cancer. As detailed below, these peptides all
are either 29 or 35 amino acids in length and are substantially homologous
in amino acid sequence.
A. Purification
The purification of the SCM-recognition factor to substantial homogeneity
from blood plasma was performed as described in U.S. patent application
Ser. No. 07/167,007 by Drs. Boris and Lea Cercek, entitled "General
Cancer-associated SCM-recognition Factor, Preparation and Method of Use"
and incorporated herein by this reference. The purification process
preferably occurs in five steps: (1) ultrafiltration; (2) desalting; (3)
gel filtration; (4) anion-exchange chromatography; and (5) reverse-phase
high-pressure liquid chromatography (RP-HPLC).
1. Ultrafiltration
The first step in purification of the SCM factor is obtaining an
ultrafiltrate from a body fluid of a donor afflicted with cancer. The body
fluid can be peripheral blood, blood plasma, or urine; if the fluid is
peripheral blood, the blood is centrifuged to separate the red blood cells
from the plasma. The donor of the body fluid used for isolation of the SCM
factor can be either autologous or allogeneic with respect to the
lymphocytes used for the SCM test. Alternatively, the SCM factor can be
purified from cell aspirates or other cellular materials derived from
patients with malignancies.
The ultrafiltration process separates the first fraction of the body fluid
comprising molecules having an apparent molecular weight greater than
1,000 daltons from a second fraction comprising molecules having an
apparent molecular weight less than 1,000 daltons. The general
cancer-associated SCM factor of the present invention is found in the
second fraction of the ultrafiltrate. The terms "apparent molecular
weight" and "nominal molecular weight cutoff" are used herein because
ultrafiltration is a somewhat imprecise method of separating molecules
according to molecular weight in this molecular weight range, and the
exact molecular weight excluded by a filter with a nominal molecular
weight cutoff of 1,000 daltons depends somewhat on the conformation of the
molecule. Molecules larger than 1,000 daltons in actual molecular weight
can, in fact, pass through an ultrafilter with a nominal molecular weight
cutoff of 1,000 daltons if, for example, the molecules are relatively long
and narrow. In fact, the purified general cancer-associated SCM factors of
the present invention are either 29 or 35 amino acids long and have
molecular weights of approximately 3,200 or 3,900 daltons, respectively.
Nevertheless, all of these peptides pass through an ultrafilter with a
nominal molecular weight cutoff of 1,000 daltons.
Preferably, the separation of the second fraction from the first fraction
is performed by filtration of the body fluid through an ultrafilter with a
nominal 1,000-dalton molecular weight cutoff, such as, but not limited to,
an Amicon.TM. UM2 or YM2 filter (available from Amicon Corporation,
Scientific System Division, Danvers, Mass. 01923).
The purity of a preparation of such a factor, at the ultrafiltrate stage or
later, can be described by its specific activity. In this context, the
term "specific activity" is defined as the reciprocal of the quantity of
protein required to cause a particular degree of decrease, such as 20%, in
the intracellular fluorescence polarization value when a particular
fraction is used to challenge SCM-responding lymphocytes in the SCM test.
The goal of purification of the SCM factor is to increase the specific
activity of the SCM factor over the specific activity found in the crude
ultrafiltrate. The process of purification can therefore be followed by
determining the specific activity of the purified fractions at each stage.
Since the protein concentration in the examples reported herein is only
determined approximately in terms of ultraviolet absorbance, preferably at
220 nm, and the complete dose-response curve for the factor has not yet
been determined, the characterization of various steps of the purification
of the SCM factor described herein in terms of specific activity is only
approximate. However, it is clear that the protein concentration decreases
markedly as the factor moves through the various purification steps while
the activity of the factor is relatively unaffected, thereby resulting in
an increase in specific activity of the SCM factor. Nevertheless, even the
ultrafiltrate can properly be described as consisting essentially of
substantially purified general cancer-associated SCM-recognition factor,
inasmuch as ultrafiltration through a membrane with a nominal molecular
weight cutoff of 1,000 daltons removes from a biological fluid the
overwhelming majority of molecules with any biological activity, including
all proteins and larger peptides.
2. Desalting
The next step in the purification of the general cancer-associated SCM
factor is a desalting step in which the fraction obtained from
ultrafiltration is loaded on a chromatographic column capable of
separating the salts therefrom. The material loaded onto the column is
then eluted from the column with distilled water, and the portion eluting
at an elution volume of between about 0.3 and about 0.5 times the total
chromatographic bed volume, containing the SCM factor, is collected.
Preferably, the column used in this step is a gel-filtration column with a
fractionation range of from 0 to about 700 daltons, such as Sephadex.TM.
G-10 (Pharmacia, Uppsala, Sweden), a dextran gel. A polyacrylamide gel
with corresponding separation characteristics can also be used.
3. Gel Filtration
The next step in the purification is another gel filtration step, again
separating according to size. The SCM-containing material obtained from
the desalting step is loaded onto another gel filtration column with a
fractionation range of from about 1,500 to about 30,000 daltons.
Preferably, the gel filtration column material is a dextran such as
Sephadex.TM. G-50, but a corresponding polyacrylamide gel can also be
used. The material loaded onto the column is then eluted therefrom with a
weak aqueous solution of an ammonium salt. Preferably, the ammonium salt
is ammonium bicarbonate, more preferably 50 mM ammonium bicarbonate. That
portion eluting at an elution volume between about 0.4 times and about 0.6
times the total chromatographic bed volume contains the SCM factor and is
collected.
4. Anion-exchange Chromatography
The next step in the purification is an anion-exchange chromatography step,
separating by charge. The SCM factor-containing material from the previous
gel filtration step is loaded onto an anion exchange column, preferably
diethylaminoethyl-cellulose (DEAE-cellulose). The material loaded onto the
column is then eluted therefrom with an increasing concentration of an
ammonium salt. Preferably, the ammonium salt is ammonium bicarbonate and
the increasing concentration of the ammonium salt is from 10 mM to 1.0M
ammonium bicarbonate. The fraction eluting from the column at about 0.28M
to 0.31M ammonium bicarbonate contains the SCM factor and is collected.
5. Reverse-phase High-pressure Liquid Chromatography
The final step of purification is reverse-phase high-pressure liquid
chromatography (RP-HPLC), which separates by charge and/or hydrophobicity.
Typically, the SCM factor-containing material from the DEAE-cellulose
column eluate is loaded onto an Aquapore.TM. RP-300 RP-HPLC column with
dimensions of 220 mm.times.2.1 mm. Elution is then performed with a
combination of two solvents: initially, 90 volume percent of 0.1 volume
percent aqueous trifluoroacetic acid (TFA) (solvent A) and 10 volume
percent of 0.09 volume percent of TFA in aqueous 70% acetonitrile (solvent
B), followed by a gradient with an increasing concentration of solvent B.
The SCM factor from all starting materials elutes as an homogeneous peak
at a solvent composition of 26 volume percent solvent A and 74 volume
percent solvent B.
Alternatively, RP-HPLC can be performed on a Beckman Instruments
ULTRASPHERE.RTM. ODS.TM. column. With this column, elution is then
performed with a somewhat different solvent pattern, initially 70 volume
percent of solvent A and 30 volume percent of 0.1 volume percent aqueous
TFA in aqueous 70% acetonitrile (solvent C), followed by a gradient with
an increasing concentration of solvent C. The SCM factor always elutes as
an homogeneous peak at a solvent composition of 43.7 volume percent of
solvent A and 56.3 volume percent of solvent C when the ULTRASPHERE.RTM.
column and this solvent system is used.
B. Structure of the Isolated Cancer-associated SCM-recognition Factor
The amino acid sequences of the SCM factors isolated from blood plasmas
from patients with 12 different types of cancer have been determined by
sequential Edman degradation and the results reported in Example 14.
Certain residues are unidentified; these residues are likely cysteine and
are reported herein as such. In nine out of the twelve cancers, the SCM
factor was 29 amino acids long; in the remaining three, an additional six
amino acids were present, yielding a total of 35 amino acids. In seven of
twelve of the factor preparations, polymorphisms exist, in that there are
conservative substitutions at one or two positions of the peptide. In
these cases, the preparation contains two amino acids as identified by
Edman degradation at one or two positions of the peptide. There were never
more than two such substitutions. Also, in two cases, gastric sarcoma and
prostate cancer, the SCM factor appears in two forms, one of 29 amino acid
residues and the other of 35 amino acid residues. No forms of intermediate
length are found. For seminoma of the testes, only the 35 amino acid form
is found. These slight differences in amino acid sequence do not affect
the cross-reactivity of the factors in the SCM test.
One region of the sequence is nearly invariant--residues 14-22. This
sequence is F-L-M-I-D-Q-N-T-K, except in the factors for prostate cancer
and seminoma of the testes, in which E (glutamate) replaces D (aspartate)
at position 18. This change is extremely conservative, inasmuch as
glutamate and aspartate have the same charge and differ by only one methyl
group. This region is believed to be extremely significant for the
functioning of the SCM factor, as discussed below.
C. Properties of the Isolated, Purified General Cancer-associated
SCM-recognition Factor
1. Activity in the SCM Test
The purified SCM factors are fully active in the SCM test when used as a
challenging agent for lymphocytes isolated from patients with several
different types of malignancies. This activity can be demonstrated by
assay at any point during the purification of the factor, starting at the
ultrafiltrate. Details of the results of such assays are given below under
"Examples." The greatest activity is obtained with material taken from the
final RP-HPLC step. One-tenth milliliter of this fraction, having an
approximate protein content of 40 picomoles of peptide, causes a decrease
in intracellular fluorescence polarization of as much as 44.6% when used
to challenge SCM-responding lymphocytes isolated from cancer patients, but
causes no decrease in intracellular fluorescence polarization when used to
challenge the same population of lymphocytes isolated from healthy donors.
2. Tryptic Peptides of the Factors
Purified preparations of the SCM factor from plasma of patients with lung
cancer and breast cancer were subjected to tryptic digestion, followed by
purification of the tryptic peptides by RP-HPLC. In each case, a
particular fragment eluted at 30.4 volume percent of solvent A and 69.6
volume percent of solvent B, in RP-HPLC using the Aquapore RP-300 column.
These fractions were found, by sequence analysis, to be the fragment of
the SCM factor consisting of residues 8-22. (In both cases, residue 7 is
lysine, and trypsin is known to cleave after lysine residues.)
These tryptic peptides are fully active in the SCM test (Example 10).
Approximately 5.times.10.sup.-2 femtograms of the tryptic peptide from the
SCM factor isolated from plasma from patients with lung cancer (the lung
cancer SCM factor), which is approximately 16,000 molecules, gave full
activity in the SCM test when used as challenging agent for lymphocytes
from donors with cancer. The fragment from the lung cancer SCM factor
reacted equally well with lymphocytes from donors with lung cancer and
breast cancer, but caused no response in the SCM test when used to
challenge lymphocytes from normal donors. Further details are given below
under "Examples." Significantly, both tryptic fragments include the nearly
invariant region of the peptide from amino acids 14-22.
3. Cross-reactivity of the SCM Factor
The isolated factor of the present invention is designated as a general
cancer-associated SCM-recognition factor because lymphocytes isolated from
donors with all types of cancer respond to all preparations of the factor
in the SCM test. The type of cancer afflicting the donor of the
lymphocytes need not be the same as the type of cancer afflicting the
donor of the body fluid from which the SCM factor was purified (Example
11).
4. Modification of the SCM Response by the General Cancer-associated SCM
factor
The isolated general cancer-associated SCM factor has a property of being
able to modify the response of potentially SCM-responding lymphocytes
obtained from donors free of malignancy when those lymphocytes are
contacted with the factor. Before contact, lymphocytes from donors free of
malignancy respond only to mitogens, such as phytohaemagglutinin,
concanavalin A, and pokeweed mitogen, in the SCM test and do not respond
to cancer-associated factors. However, after prolonged contact with the
SCM factor, the SCM response of the cells is modified to respond only to
cancer-associated factors and not to mitogens. In other words, contact by
such lymphocytes with the SCM factor alters their response in the SCM test
from the normal response of lymphocytes from donors free of malignancy to
the response seen with lymphocytes from donors afflicted with cancer.
Details on the demonstration of the modification of the SCM response are
given under "Examples."
5. Effect of SCM Factor on Natural Cytotoxicity of Lymphocytes
The SCM factor has a property of irreversibly suppressing the in vitro
spontaneous, natural cytotoxicity not only of the density-specific
SCM-responding subpopulation of lymphocytes, but also of the general
population of peripheral blood lymphocytes isolated by conventional
techniques. The suppression of cytotoxicity by synthetic SCM factor is
dose-dependent; only 11.5 femtomoles of the SCM factor is required for a
50% decrease of the cytotoxic effect. The suppression of cytotoxicity by
SCM factor only requires a portion of the synthetic SCM molecule, and the
region of the SCM factor responsible for the suppressive activity has been
determined. It is believed that the SCM factor is involved in the defense
of cancer cells against the attack of killer lymphocytes. This defense is
believed to help the survival and unrestrained growth of cancer cells. The
importance of the normal functioning of the immune system in controlling
the growth of cancer cells is seen by the frequent occurrence of unusual
forms of cancer in patients undergoing immune suppression. Such immune
suppression can occur as a result of a disease such as Acquired
Immunodeficiency Syndrome (AIDS) or as a result of the administration of
immunosuppressive drugs to prevent rejection of transplants. An important
example of such an unusual form of cancer is the occurrence of aggressive
forms of Kaposi's Sarcoma, ordinarily a slowly-spreading and rarely fatal
cancer, in AIDS patients. Details on the decrease of natural toxicity of
lymphocytes are given below under "Examples."
However, this immunosuppressive effect of the SCM factor could in some
instances be beneficial to a patient without cancer. For example, in
patients receiving tissue transplants and at risk of rejection of the
transplants, suppression of the cytotoxic action of lymphocytes by SCM
factor and/or its active portion could help to prevent rejection of the
transplants.
6. Homology with .alpha.-1-Protease Inhibitor
Computer search of the National Biomedical Research Foundation protein
sequence data bank unexpectedly revealed that the amino acid sequences of
the 12 isolated and purified general cancer-associated SCM-recognition
factors are from 82.8% to 89.7% identical to an internal 28-33 amino acid
sequence from the glycoprotein .alpha.-1-protease inhibitor
(.alpha.-1-PI). The .alpha.-1-PI is a glycoprotein with a molecular weight
of 55,000 daltons; it is a single polypeptide chain of 394 residues, and
inhibits serine proteases. The sequence of the .alpha.-1-PI homologous to
the SCM factor is, for factors from 9 out of 12 cancers, between amino
acids 358 and 388 with serine at position 359 missing. For the remaining
three cancers, gastric cancer, adenocarcinoma of the prostate, and
seminoma of the testes, the homologous sequence is between residues 359
and 393. For the factor from seminoma testes, the homology is 100%; for
the factor from prostate adenocarcinoma, the homology is 97%; and for the
factor from gastric carcinoma, the homology is 94%. (These calculations
exclude the unidentified residues.)
In the SCM factors identified from 9 out of 12 types of cancer, the
amino-terminal residue is either methionine (5 cancers), or valine (4
cancers); in two additional factors, it is arginine. In 11 out of the 12
SCM factors, the amino acid serine, originally at position 359, next to
the methionine and the active site of .alpha.-1-PI at position 358, is
missing. In the seminoma testes SCM factor, the serine is present at the
amino-terminal position, but methionine is absent.
The .alpha.-1-PI is a glycoprotein normally synthesized in the liver and
rapidly released in the blood plasma. Normal levels of this glycoprotein
in plasma are reported to be 1.3 g/l. It is an acute-reactive protein and
its synthesis increases up to 4-fold in response to inflammatory signals
and other homeostatic needs. It inhibits serine proteases and plays an
important role in inflammatory processes by defending tissues against
attack of proteolytic enzymes released by leukocytes at the site and
source of inflammation. It is also thought to be part of the regulatory
mechanisms of DNA synthesis, the cell division cycle, and differentiation
and maturation processes. Inadequate protease inhibition unbalances these
processes, often with deleterious consequences to the host. On the other
hand, the absence of protease inhibition increases the fertilization
efficiency, possibly promoting propagation of individuals with
.alpha.-1-PI deficiencies. It has been suggested that the level or type of
.alpha.-1-PI may influence pathophysiological processes and determine the
occurrence, course, and severity of disease.
However, no genetic variants of .alpha.-1-PI are known that could account
for the presence of SCM factors in blood plasma, either as a product of an
aberrant cleavage at the active center during an inhibition reaction with
a protease of a cancer cell, or as a defect in synthesis.
There is also no evidence that peptides similar to the SCM factors are
generated as a result of breakage caused by ultrafiltration. Plasma from
donors free of cancer, including plasma from patients with inflammatory
diseases, was subjected to the same ultrafiltration process used as the
first stage in the purification process of SCM factor. No fragments of
.alpha.-1-PI or peptides similar to the SCM factor were detected in the
ultrafiltrates. Furthermore, SCM-responding lymphocytes from cancer
patients did not respond in the SCM test to such ultrafiltrates.
To eliminate the possibility that some specific, aggressive proteases
secreted by tumor cells could cleave .alpha.-1-PI to produce molecules
similar to SCM factors, we incubated overnight at 37.degree. C. a variety
of human tumor biopsies and human cultured cancer cell lines in the
presence of: (a) pure human .alpha.-1-PI; (b) a complex of trypsin and
.alpha.-1-PI; and (c) the molecular weight fraction of cancer patients'
plasmas above 5,000 daltons containing .alpha.-1-PI, to ascertain that no
labile genetic variant of .alpha.-1-PI is present in the plasma of cancer
patients. After incubation, tissues or cells were separated by
centrifugation and the supernatants were subjected first to
ultrafiltration through 1,000-dalton molecular weight cutoff filters
(Amicon.TM. YM2) and then through further chromatographic procedures as
used in the purification of SCM factors. None of these preparations
yielded any SCM factor in quantities above those measured in supernatants
of untreated control cancer cells.
7. Synthesis of SCM Factors by Cancer Cells in Culture
Metabolically active human cancer cells grown in culture, including T10806
fibrosarcoma cells, MCF7 breast cancer cells, A2780 ovarian cancer cells,
and HCT80 colon cancer cells, excreted into serum-free tissue culture
media molecules that, when taken through the SCM factor purification
process, exhibited optical density peaks with retention times identical to
those for SCM factor itself.
Sequencing of the picomolar amounts of SCM factor present in the purified
preparations of SCM factor from supernatant medium in which the human MCF7
breast cancer cells and HCT80 colon cancer cells were grown confirmed that
cells grown in vitro excrete molecules homologous with SCM factor. As
shown in Example 24, 15 of the first 16 amino acid residues in the
preparation from MCF7 breast cancer cells and 5 of the first 6 amino acid
residues in the preparation from HCT80 colon cancer cells were identical
to the sequence obtained from the SCM factor purified from plasma of
breast and colon cancer patients, respectively.
These results were supported by ELISA tests using anti-SCM factor antibody
(Example 25). When ELISA tests were performed on the cultured human cancer
cells, the presence of SCM factor was detected in all of the cell lines
tested. Different cell lines produced different quantities of SCM factor
per cell under identical conditions. This variation might be an expression
of differences in carcinogenic potential or metabolic activity of these
different cell lines. This is supported by results showing the treatment
of MCF7 breast cancer cells and T1080 fibrosarcoma cells with
cycloheximide, a translational inhibitor of protein synthesis, caused a
decrease in the synthesis of SCM factor. These results are in agreement
with our hypothesis that cancer cells actively synthesize SCM factor
molecules.
To eliminate the possibility that supernatant growth media and/or cultured
cells would be contaminated by some variants of an .alpha.-1-PI produced
by fetal cells, fetal calf serum was omitted from the growth media used
for the last two medium changes. Additionally, we have subjected fetal
calf serum to ultrafiltrations through filters with a cutoff of 1,000
daltons followed by the same chromatographic procedure used for the
purification of SCM factor. The RP-HPLC eluate resulting did not show an
optical density peak at the retention time characteristic of SCM factor
molecules. However, another peak adjacent to that for SCM factor was
collected in sequence. There was a non-linear sequence homology of 44.7%
of the amino acids present between this peak and SCM factor that could
indicate a similar genetic origin, but the difference in sequence is too
large to justify any conclusion that SCM factor, like
.alpha.-fetoproteins, is a product of cancer cell dedifferentiation. This
suggests that SCM factor is not an ectopic, dedifferentiation tumor
marker. More importantly, the SCM factor itself was not present in the
fetal calf serum.
Active protein synthesis is required for production of SCM factor by cancer
cells. Example 26 shows that treatment of cultured human cancer cells with
the protein synthesis inhibitor cycloheximide considerably decreased the
synthesis of the SCM factor as determined by the ELISA assay. Similarly,
Example 30 shows that addition of ascorbate ions to cultures of MCF7
breast cancer cells considerably decreased the synthesis of SCM factor by
the ELISA assay. Since ascorbate ions can inhibit protein synthesis by
reverting mitochondria of cancer cells into the idle, orthodox
conformation, as described in L. Cercek & B. Cercek, "Effect of Ascorbate
Ions on Intracellular Fluorescein Emission Polarization Spectra in Cancer
and Normal Proliferating Cells," Cancer Detection & Prevention 10, 1-20
(1987), these results confirm that active protein synthesis is required
for production of SCM factor.
8. Stimulation of DNA Synthesis by SCM Factor
As shown in Example 27, SCM factor enhances DNA synthesis of rat
hepatocytes grown in culture, as determined by tritiated thymidine uptake.
The enhancement of DNA synthesis is dependent on the dose of SCM factor
administered. The relationship between the activity of SCM factor in
stimulating DNA synthesis and its possible role in promoting the growth of
cancer cells is discussed below.
9. Blockage of .alpha.-1-PI Activity
SCM factor has no inhibitory or inactivating activity against serine
proteases, unlike .alpha.-1-PI. However, SCM factor can block the
inhibitory or inactivating activity of .alpha.-1-PI on proteases when SCM
factor molecules are added to the protease before or simultaneously with
the .alpha.-1-PI. This might result in a possible increase of protease
activity in cancer cells producing SCM factor. The possible consequences
of this are discussed below.
II. SYNTHETIC CANCER-ASSOCIATED SCM-RECOGNITION FACTOR
In view of the high degree of sequence homology between the SCM factors
isolated from 12 different types of cancer, a synthetic SCM factor has now
been prepared using standard solid-phase peptide synthesis methods. This
synthetic SCM factor has a "consensus" sequence of 29 amino acids and
shares the properties and activity of the isolated purified SCM factors.
The preparation of a synthetic SCM factor is desirable for a number of
reasons: (1) availability and quantity without the necessity of isolation
from cancer tissues; (2) uniformity of structure and activity; and (3) the
possibility of varying the sequence in order to determine
structure-activity relationships.
A. Sequence of the Synthetic SCM Factor Molecule
The synthetic SCM factor has the amino acid sequence
M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K.
This sequence is not the only sequence with 29 amino acids believed to
possess SCM activity. It is a well-established principle of protein and
peptide chemistry that certain amino acids substitutions, entitled
"conservative" amino acid substitutions, can frequently be made in a
protein or a peptide without altering either the confirmation or the
function of the protein or peptide. Such changes include substituting any
of isoleucine (I), valine (V), and leucine (L) for any other of these
amino acids; aspartic acid (D) for glutamic acid (E) and vice versa;
glutamine (Q) for asparagine (N) and vice versa; and serine (S) for
threonine (T) and vice versa.
In view of these likely equivalencies, peptides of the sequence M-X.sub.2
-P-P-X.sub.5 -X.sub.6 -K-F-X.sub.13 -F-X.sub.15 -M-X.sub.17 -X.sub.18
-X.sub.19 -X.sub.20 -X.sub.21 -K-X.sub.23 -P-X.sub.25 -F-M-G-L, in which:
X.sub.2, X.sub.6, X.sub.13, X.sub.15, X.sub.17, X.sub.23, and X.sub.25 can
each be I, L, or V; X.sub.5 and X.sub.18 can each be D or E; X.sub.9,
X.sub.19 and X.sub.20 can each be Q or N; and X.sub.21 can be S or T, by
definition are expected to have SCM factor activity. In this designation
of the sequence, and corresponding designations elsewhere employing
subscripts, the number appearing in the subscript indicates the position
of the amino acid specified in a factor of 29 amino acids. For example,
"X.sub.2 " refers to the second amino acid from the amino-terminus.
The above-mentioned substitutions are not the only amino acid substitutions
that can be considered "conservative." Other substitutions can also be
considered conservative, depending on the environment of the particular
amino acid. For example, glycine (G) and alanine (A) can frequently be
interchangeable, as can be alanine and valine (V). Methionine (M), which
is relatively hydrophobic, can frequently be interchanged with leucine and
isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are
frequently interchangeable in locations in which the significant feature
of the amino acid residue is its charge and the differing pK's of these
two amino acid residues are not significant. Still other changes can be
considered "conservative" in particular environments.
B. Properties of the Synthetic SCM Factor Activity in the SCM Test
The synthetic SCM factor molecule is highly active in the SCM test. As
shown below in Example 15, as little as 2 femtomoles (2.times.10.sup.-15
moles) of the synthetic SCM factor molecule produced a significant, 20%,
decrease in intracellular fluorescence polarization in the SCM test when
used to challenge SCM-responding lymphocytes. The synthetic peptide is
active in the SCM test when used to challenge SCM-responding lymphocytes
from donors with tumors of different histological type and in different
organs. The corresponding fraction of SCM-responding lymphocytes from
normal, healthy donors does not respond to the SCM factor in quantities as
large as 960 picomoles (960.times.10.sup.-12 moles).
2. Induction of SCM-recognition Receptors in Lymphocytes from Healthy
Donors
The synthetic SCM factor can modify the SCM response of lymphocytes from
healthy donors from the response characteristic of such lymphocytes (i.e.,
a response to PHA and no response to a cancer-associated factor) to the
response characteristic of lymphocytes from donors with cancer (i.e., no
response to PHA and a response to a cancer-associated factor). As detailed
in Example 19, SCM-responding lymphocytes from healthy donors did not
respond to synthetic SCM factor in the SCM test. However, after incubation
for 2.5 hours at 37.degree. C. in the presence of 400 picomoles of
synthetic SCM factor per 5.times.10.sup.6 cells, followed by three washes
with phosphate-buffered saline (PBS), these cells showed a 37% decrease in
intracellular fluorescence polarization, indicating the induction of
receptors that can respond to the synthetic SCM factor.
The induction of these receptors requires protein synthesis. When the
incubation is carried out in the presence of the protein synthesis
inhibitors cycloheximide or actinomycin D at 10 .mu.g/5.times.10.sup.6
cells, no response to synthetic SCM factor was induced, and the normal
response to the mitogen PHA was not abolished.
3. Immunogenic Properties of Synthetic SCM Factor
The synthetic SCM factor has a predominantly .alpha.-helical secondary
structure and is large enough to suggest that it could be presented by the
major histocompatibility complex (MHC complex) for induction of the immune
response. To test this assumption, the synthetic SCM factor was used to
immunize experimental animals, as detailed below in Example 20. Both pure
synthetic SCM factor and synthetic SCM factor conjugated to keyhole limpet
hemocyanin (KLH) were used for immunization. In the latter case, the
synthetic SCM factor was conjugated to the KLH via an added carboxy
terminal cysteine residue on the SCM factor using N-succinyl bromoacetate
as the cross-linking agent.
Other carriers, such as polylysine, can also be used for immunization. The
use of such carriers is well-known in the art.
4. Effect of Synthetic SCM Factor and Fragments Thereof on Natural
Cytotoxicity of Lymphocytes
The synthetic SCM factor was shown to suppress the natural cytotoxic
activity of lymphocytes against cancer cells (Example 31). The synthetic
SCM factor decreased the natural killing (NK) efficiency of lymphocytes
from normal healthy donors against K562 human myeloma target cells by 97%
to 99.9% when a dose of 35 femtomoles of synthetic SCM factor was used per
lymphocyte. A dose of 11.5 femtomoles per lymphocyte resulted in a 50%
decrease in cytotoxicity (Table 23). Such quantities of SCM factor are
expected to be present in the immediate surrounding of metabolically
active cancer cells, which produce these molecules. This NK-suppressive
effect is irreversible and cannot be removed by a thorough 3-times washing
of the treated lymphocytes.
To find out to which part of the amino acid sequence of the synthetic
SCM-factor molecule this NK-suppressive activity can be ascribed, we have
tested various synthetic peptide fragments of the synthetic SCM-factor
molecule. As can be seen from Example 31 (Table 26), the NK-suppressive
effect was found only in the entire synthetic SCM factor (29 amino acids)
and fragment F2 (amino acids 8-29). None of the fragments that did not
contain the carboxyl-terminal region of the synthetic SCM factor, i.e.,
fragments F1, F3, F4, and F5 (Table 26) was active, which might indicate
that the seven carboyxl-terminal amino acid residues are responsible for
the suppression of NK activity of lymphocytes. However, the peptide
fragment consisting of these seven carboxyl-terminal residues, residues
23-29 (fragment F8) did not suppress NK activity, and neither did peptide
fragment F7, consisting of residues 14-29. This shows that the shortest
sequence possessing NK-suppressive activity resides within the portion of
the SCM factor molecule encompassing residues 8-29, and only the first
seven amino-terminal residues are not important for NK-suppressive
activity. This is in contrast to the portion of the synthetic SCM-factor
molecule that is responsible for protection against inhibition by
.alpha.-1-PI, which is the first seven amino-terminal residues of the
molecule, or the portion of the molecule responsible for SCM activity
itself, which occurs within the portion of the molecule between amino acid
residue 14 and amino acid residue 22. Thus, the SCM factor exerts multiple
functions in promoting the growth and invasion of cancer cells and in
suppressing host defenses.
These results indicate that for NK suppressive activity to be expressed,
the peptide need not contain the first seven amino-terminal residues of
the synthetic SCM factor. Fragment F2 has the amino acid sequence
F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K. As explained above, variants
of fragment F2 containing conservative amino acid substitutions are also
expected to have NK-suppressive activity.
Furthermore, as can be seen from the results in Example 31, the
NK-suppressive action of synthetic SCM factor not only affects the
density-specific, SCM-responding subpopulation of lymphocytes, but a wider
population spectrum of lymphocytes as isolated from peripheral blood by
using conventional density-gradient centrifugation techniques employing
the Histopaque density medium. This indicates that the effect of synthetic
SCM factor on suppression of lymphocyte cytotoxicity has a broad spectrum
of action. This broad spectrum of action includes various mechanisms by
which the SCM factor can inhibit the cytotoxicity of lymphocytes against
malignant cells. For example, we have observed that complex formation
between the lymphocytes and K562 myeloma cells, one of the first steps in
the killing process, is decreased when lymphocytes are treated with the
SCM factor. Other effects of the SCM factor could be prevention of
synthesis of leukolysins and/or direct inactivation of various cytolytic
molecules, such as tumor necrosis factor (TNF).
The NK-suppressive activity of SCM factor or portions of SCM factor can be
used to assess the effectiveness of an anti-cancer agent capable of
inhibiting the growth of malignant cells in a cell culture that includes
both lymphocytes exhibiting NK activity and malignant cells. The
effectiveness can be assessed by:
(1) incubating the cell culture with a substantially purified
NK-suppressive peptide, such as intact SCM factor or one of the peptides
expected to have NK-suppressive activity described above, in a quantity
sufficient to substantially suppress the NK activity of the lymphocytes of
the cell culture;
(2) adding the anti-cancer agent to the cell culture in a quantity
sufficient to measurably inhibit the growth of the malignant cells; and
(3) determining the effect of the anti-cancer agent on the malignant cells
by observing the inhibition of growth of the malignant cells caused by the
anti-cancer agent in the essential absence of NK activity caused by the
lymphocytes.
C. Production and Activity of Fragments of Synthetic SCM Factor
1. Sequences and Activity of Fragments
In order to determine which portion or portions of the synthetic SCM factor
is responsible for its activity in the SCM test, five peptide fragments of
the synthetic SCM factor were synthesized, designated F1 through F5. These
represented the following portions of the intact molecules: F1, amino
acids 1-22; F2, amino acids 8-29; F3, amino acids 8-22; F4, amino acids
14-22: and F5, amino acids 1-13. These fragments have the following amino
acid sequences:
F1: M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-D-Q-N-T-K;
F2: F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K;
F3: F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K;
F4: F-L-M-I-D-Q-N-T-K; and
F5: M-I-P-P-E-V-K-F-N-K-P-F-V-F.
As detailed below in Example 17, fragments F1, F2, F3, and F4 are all
active in the SCM test, while fragment F5 is inactive. All of the active
fragments contain the 9-amino-acid segment of F4, and it is reasonable
that this segment might represent the active site responsible for SCM
activity.
Not only are peptides F1 through F4 active in the SCM test, variants of
these peptides with conservative amino acid substitutions are also
expected to have SCM activity and fall within the scope of the present
invention. These conservative substitutions, as outlined above, include
any of isoleucine, valine, and leucine for any other of these amino acids;
aspartic acid for glutamic acid and vice versa; asparagine for glutamine
and vice versa; and serine for threonine and vice versa. The existence of
these conservative substitutions means that the following peptides are
expected to have SCM activity:
M-X.sub.2 -P-P-X.sub.5 -X.sub.6 -K-F-X.sub.9 -K-P-F-X.sub.13 -F-X.sub.15
-M-X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20 -X.sub.21 -K;
F-X.sub.9 -K-P-F-X.sub.13 -F-X.sub.15 -M-X.sub.17 -X.sub.18 -X.sub.19
-X.sub.20 -X.sub.21 -K-X.sub.23 -P-X.sub.25 -F-M-G-K;
F-X.sub.9 -K-P-F-X.sub.13 -F-X.sub.15 -M-X.sub.17 -X.sub.18 -X.sub.19
-X.sub.20 -X.sub.21 -K; and
F-X.sub.15 -M-X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20 -X.sub.21 -K.
In these sequences, the subscripts designating particular amino acid
residues have the same meaning as stated above in the discussion of
conservative amino acid substitutions in the entire 29-amino-acid
synthetic SCM factor.
2. Use of the Amphipathicity Profile to Determine SCM Activity
An amphipathicity profile is a plot of the relative hydrophilicity or
hydrophobicity of segments of a peptide or protein. Amino acid residues
range from quite hydrophilic (e.g., charged residues or serine) to quite
hydrophobic (e.g., phenylalanine). Typically, the plot is presented as a
moving average over a short stretch of amino acids within the protein or
peptide. For specificity and recognition purposes, amphipathicity
properties of short peptides can be as significant as the amino acid
sequence itself. As shown below in Example 18, the amphipathicity profile
of the SCM-active F4 peptide fragment is strikingly similar to the
amphipathicity profile of the synthetic 8-amino-acid peptide with
SCM-factor activity having the sequence of F-W-G-A-E-G-Q-R, even though
there is only a limited sequence homology between this peptide and the F4
peptide. By contrast, the experimental allergic encephalitogenic peptide
(EAE peptide) has a sequence of F-S-W-G-A-E-G-Q-R. The presence of the
relatively hydrophilic serine between the hydrophobic residues
phenylalanine and tryptophan alters the amphipathicity profile
considerably. As detailed in our prior U.S. patent application Ser. No.
07/167,007, the EAE peptide has no SCM factor activity.
Given the importance of the amphipathicity profile of a peptide in
determining whether the peptide has SCM factor activity, a peptide of at
least 9 amino acid residues including a core sequence of 9 amino acid
residues having an amphipathicity profile substantially equivalent to that
of the sequence F-L-M-I-D-Q-N-T-K is expected to have SCM factor activity.
III. USE OF THE PURIFIED AND SYNTHETIC SCM FACTORS
Both the purified and synthetic SCM factors can be used as challenging
agents in the SCM test, can be used to prepare antisera for the detection
of the SCM factor, and can be used for the generation of DNA sequences
that carry equivalent genetic information for use in a variety of genetic
engineering procedures. As discussed below, this SCM factor can also be
used in the management of cancer.
A. Performance of the SCM Test
The activity of both the purified SCM factor and the synthetic SCM factor,
as well as the fragments of the SCM factor, is confirmed by its effect on
viable SCM-responding lymphocytes in accordance with the prior publication
by L. Cercek and B. Cercek, "Application of the Phenomenon of Changes in
the Structuredness of Cytoplasmic Matrix (SCM) in the Diagnosis of
Malignant Disorders: A Review," Europ. J. Cancer 13, 903-915 (1977). The
general cancer-associated SCM-recognition factor of the present invention
produces a significant decrease in the intracellular fluorescence
polarization value of potentially SCM-responding lymphocytes from donors
afflicted with cancer when used to challenge such lymphocytes in the SCM
test as performed as described in that article. The degree of decrease of
the intracellular fluorescein fluorescence polarization value of such
challenged lymphocytes is substantial--at least 20% even if ultrafiltrate
from plasma from donors afflicted with cancer is used to challenge such
lymphocytes, and as great as 40-55% if purified RP-HPLC fractions or
synthetic peptides are used.
Two previously established procedures are important for the proper
performance of the SCM test as reported herein. These procedures are the
isolation of potentially SCM-responding lymphocytes and the technique of
measuring the fluorescence polarization values themselves, and their
conversion into numbers meaningful for the SCM test.
1. Isolation of SCM-responding Lymphocytes
Several procedures for the isolation of potentially SCM-responding
lymphocytes are described in the European Journal of Cancer review
article, supra, and also in a prior patent application by B. Cercek and L.
Cercek, U.S. application Ser. No. 07/260,928, filed Oct. 21, 1988, and
entitled "Provision of Density Specific Blood Cells for the Structuredness
of the Cytoplasmic Matrix (SCM) Test," incorporated herein by this
reference. The separation of these lymphocytes from the general lymphocyte
population is important for the proper performance of the SCM test,
because only a relatively small portion of lymphocytes, approximately
20-25% of the total lymphocyte population, is capable of responding in the
SCM test to cancer-associated factors. Therefore, to perform the test on
unfractionated lymphocytes results in a much smaller observed decrease in
the intracellular polarization value even when the lymphocytes actually
capable of responding in the SCM test fully respond to the challenging
agent used. Additionally, there exists another fraction of lymphocytes
that responds in the SCM test in a different way. These cells do not
respond at all to cancer-associated factors, but respond to PHA when
isolated from donors with cancer. As detailed below, some variations of
the SCM test compare the response to PHA to a cancer-associated factor,
such as a peptide of the present invention, to determine whether or not
the donor of the lymphocytes is afflicted with a malignancy. This second
fraction of lymphocytes therefore must be rigorously excluded to avoid
distorting the results.
Immunologically, the SCM-responding lymphocytes are T-cell mononuclear
leukocytes. Although not fully understood, it is believed that
SCM-responding lymphocytes are involved in the recognition of antigens
that are circulating in the blood stream, expressed on cancer cells, or
excreted by cancer cells into interstitial spaces of tumors. This
recognition of antigens triggers the body's immune system. Accordingly,
these cells become primed to recognize foreign substances, such as
antigens, produced by the disease or condition affecting the body.
SCM-responding lymphocytes can be isolated by using either a single-density
solution, a step gradient, or a continuous preformed gradient.
a. Isolation Using Single-density Solution
For the isolation of SCM-responding lymphocytes by the single-density
solution method, a sample of peripheral blood is drawn from a donor and
collected in a heparinized tube. After collection, the peripheral blood is
treated with iron powder or carbonyl-iron powder and the tubes containing
the blood-iron powder mixture are placed on a magnet to effect separation
of the phagocytic cells along with the iron powder from a blood sample. A
portion of the blood sample depleted of phagocytic cells is then
transferred to a Ficoll.TM.-Triosil.TM. density-gradient solution and
centrifuged to effect separation of the SCM-responding lymphocytes based
on density differences. This method of separation typically uses a
density-gradient solution having a density of 1.081 g/cm.sup.3 at
25.degree. C. and an osmolality of 0.320 Osm/ kg. Centrifugation is
carried out at 550.times.g for 20 minutes at a temperature of 25.degree.
C. The SCM-responding lymphocytes are recovered using a Pasteur pipette to
remove the cell layer separated above the density-gradient material.
Removal of the density-gradient material must be avoided as far as is
possible as this material includes various heavier plasma and cell
components which interfere with the test results. Removal of the lighter
plasma material should also be avoided as much as possible to eliminate
the introduction of any contaminating components or SCM-nonresponding
cells into the test samples.
Following separation, the SCM-responding lymphocytes are subjected to
several washing steps, first in 0.9% preservative-free sodium chloride
solution, then in complete Dulbecco's phosphate-buffered saline (PBS) and
held at 37.degree. C. for subsequent use in the SCM test procedure.
b. Isolation Using Step Gradient
For the isolation of SCM-responding lymphocytes using a step gradient, use
a total blood sample depleted of phagocytic cells as described above, or
the total population of peripheral blood lymphocytes can be used. The use
of the total population of blood lymphocytes is preferred, as this avoids
the use of possibly toxic iron or carbonyl-iron powder.
The isolation of the total population of peripheral blood lymphocytes for
an heparinized blood sample is also performed using density-gradient
centrifugation. This centrifugation step is performed by layering the
heparinized blood on top of a solution of density 1.077 g/cm.sup.3
containing a non-ionic synthetic polymer of sucrose with a molecular
weight of about 400,000 and sodium diatrizoate. Both solutions are
equilibrated to room temperature, and the volume of the heparinized blood.
The layered solutions are then density solution is at least as great as
the volume of centrifuged, typically at 30 minutes at room temperature at
550.times.g, so that the lymphocytes are banded at the interface between
the solutions. The lymphocytes are then collected from the interface.
For the step of separation of the SCM-responding lymphocytes, a step
gradient is made by layering a solution of density 1.0590 g/cm.sup.3 and
osmolality of 0.320 Osm/kg on top of a solution of density 1.0670
g/cm.sup.3 and the same osmolality. These solutions are typically prepared
from polyvinylpyrrolidone-covered silica media such as Percoll.TM.
(Pharmacia, Uppsala, Sweden). A volume of a blood sample or peripheral
blood lymphocytes equal to about one-half the total volume of the step
gradient is layered on top of the step gradient, and the mixture is
centrifuged typically at 550.times.g for 30 minutes. The SCM-responding
lymphocytes collect in a visible band between the first and second density
solution and are harvested.
c. Isolation Using Continuous Preformed Gradient
As an alternative to the step gradient, a continuous preformed gradient can
be used for the final separation of SCM-responding lymphocytes. This
gradient spans a density range of between 1.050 g/cm.sup.3 and 1.070
g/cm.sup.3. It can be generated by centrifuging a solution of
polyvinylpyrrolidone-covered silica at 26,000.times.g in a 29.degree.
fixed-angle rotor or at 11,400.times.g in a 34.degree. fixed-angle rotor.
2. Performance of the SCM Test on Isolated SCM-responding Lymphocytes
a. Measurement of SCM Values
The method for measuring the fluorescence polarization values of
SCM-responding lymphocytes in the SCM test has been described in the
European Journal of Cancer review article, supra, as well as in a prior
patent application by B. Cercek & L. Cercek, Ser. No. 867,079, filed May
27, 1986, entitled "Method for Measuring Polarized Fluorescence
Emissions," incorporated herein by this reference. As described in these
references, SCM-responding lymphocytes previously separated from the test
subject's peripheral blood are incubated in sterile glass tubes at
37.degree. C. with a known concentration of either a mitogen such as
phytohaemagglutinin or a cancer-associated antigen such as the general
cancer-associated SCM-recognition factor which is the subject of the
present invention. Other mitogens than phytohaemagglutinin (PHA), such as
concanavalin A and pokeweed mitogen, have been used, but PHA is preferred
for the SCM test. This incubation is initiated by adding 0.1 ml of the
appropriately diluted mitogen or antigen to 1 ml of the cell suspension at
6.times.10.sup.6 cells/ml. The incubation is then allowed to proceed for
30-60 min.
The incubated lymphocytes are then admixed in suspension with a suitable
nonfluorogenic compound hydrolyzable intracellularly to a fluorogenic
compound, referred to hereinafter as a fluorogenic agent precursor, such
as fluorescein diacetate (FDA). The fluorescein diacetate is used at a
final concentration of 2.5 mM or 0.7 mM in complete PBS at pH 7.4 and
osmolality of 0.330 Osm/kg and is diluted from a concentrated stock
solution prepared in acetone or glacial acetic acid, respectively.
Aliquots of 0.2 ml of control or stimulated lymphocyte suspensions are
slowly injected with a syringe into a beaker containing 3 ml of the FDA
substrate solution.
The cells are exposed to the FDA for sufficient time (about 5 minutes) to
allow for the penetration of the FDA substrate solution into the
lymphocytes. Inside the cells, the nonfluorogenic fluorescein diacetate
molecules are converted to fluorescein molecules by enzymatic hydrolysis.
The fluor-containing lymphocytes are isotropic in their response to
polarized light since the polarization of the emitted fluorescence
relative to that of the exciting light does not depend on the orientation
of the plane-polarized light used to excite the lymphocytes. However, the
conventional fluorescence polarization measuring apparatus used herein for
these measurements uses vertically polarized exciting light to excite the
lymphocytes, so the measurement process is described in terms of
vertically polarized exciting light.
When exposed to excitation energy in the form of vertically polarized
light, the fluorescein molecules emit fluorescence. The relationship
between the vertically polarized and horizontally polarized emissions is
measured. This can be done by measuring the polarized fluorescence
intensities in both the vertical and horizontal planes and determining a
polarization value (P value) in accordance with the following
relationship:
##EQU2##
where I.sub.V and I.sub.H are polarized fluorescence intensities in the
vertical and horizontal planes, respectively, and G is a correction factor
for the unequal transmission of the horizontal and vertical components of
the polarized light through the optical system of the particular equipment
used. The value of G is determined by dividing the intensity of the
horizontally polarized light by the intensity of the vertically polarized
light emitted from a 10.sup.-7 M solution of fluorescein in PBS excited
with horizontally polarized light of the same wavelength as used for the
SCM measurements. For the measurements reported herein, G=0.42.
The P value of stimulated lymphocytes, that is those lymphocytes that have
been exposed to the general SCM-associated cancer recognition factor of
the present invention, is compared with the P value of a control
suspension of unstimulated lymphocytes from the same donor and the percent
decrease in P value of the stimulated lymphocytes as compared to the P
value of the control lymphocytes is an indication of the SCM-response to
the cancer antigen.
Although the SCM response can be observed through some range of excitation
and emission wavelengths, when using FDA as the fluorogenic agent
precursor, it is strongly preferred to use an excitation wavelength of 470
nm and an emission wavelength of 510 nm. All results hereinafter described
were obtained using those wavelengths. However, good results have also
been achieved using an excitation wavelength of 442 nm and an emission
wavelength of 527 nm.
The spectrophotometer utilized for SCM fluorescence measurements should be
one of high sensitivity and stability and should be able to compensate for
fluctuations in the intensity of the exciting light since the intensity of
the polarized fluorescence emissions is recorded as a function of time and
since the bulk concentration of fluorescein in the SCM measurements is
only of the order of 10.sup.-8 M to 10.sup.-9 M. Also, broad band filter
instruments are not suited for use for SCM measurements since SCM
responses can be detected only within a narrow wavelength region. The
maximum spectral slit width of the excitation monochromator should be 20
nm and the maximum spectral slit width of the emission monochromator
should be 10 nm when the excitation monochromator is set at 470 nm and the
emission monochromator at 510 nm. The spectrophotometer should also be
fitted with a thermostatically controlled cuvette holder since the
polarized fluorescent emissions are highly temperature dependent. The
spectrophotometer should also be provided with means for measuring both
the horizontal and vertical polarized components of the fluorescent
emissions.
In the examples hereinafter set forth we used a Perkin-Elmer MPF-4
spectrophotometer which was equipped with a thermostatically controlled
cuvette holder. All measurements were carried out at 27.degree. C. The
light source was a xenon lamp.
In measuring the fluorescence polarization, the intensities of the
emissions parallel to and perpendicular to the vertical exciting light
beam are recorded alternately with an automatic polarizer changer for
about 6 minutes or until the intensity of the emission perpendicular to
the vertically exciting light beam reaches 80-90% of the full scale
deflection of the recorder.
It is necessary to correct these readings for any leakage of fluorescein
from the cells and for any background of fluorescence in the substrate
solution. To perform this correction, the cells are filtered away from the
solution on a nitrocellulose filter of 0.22 .mu.m pore size mounted in an
appropriate filter head. Using the same fluorescence polarization
measurement apparatus, the fluorescence intensities parallel to and
perpendicular to the exciting light are obtained. The corrected
fluorescein intensities for the cells are then obtained by subtracting the
values obtained from the filtrate from the total fluorescence intensities
extrapolated to the half time of filtration. This extrapolation is
necessary because the background increases during the incubation because
of the leakage of fluorescein from cells and spontaneous hydrolysis of
FDA.
Alternatively, the method of compensating for background fluorescence
described in the prior patent application by the Cerceks, Ser. No.
07/222,115, filed Jul. 20, 1988, entitled "Method for Measuring
Polarization of Bathochromically Shifted Fluorescence," and incorporated
herein by this reference, can be used. Briefly, this method eliminates the
need to filter each sample by measuring the horizontally and vertically
polarized fluorescence emissions at more than one wavelength and
calculating the intracellular fluorescence emissions therefrom.
An SCM test performed according to the protocol described hereinabove,
using 1.0 ml of a lymphocyte suspension at 6.times.10.sup.6 cells/ml and
0.1 ml of the mitogen or antigen, with FDA as the fluorogenic agent
precursor and using an excitation wavelength of 470 nm and an emission
wavelength of 510 nm, is referred to herein as a "standard SCM test."
b. Interpretation of the SCM Test
The result of the SCM test is a value for the intracellular fluorescein
fluorescence polarization of the challenged lymphocytes. This value is
designated as a P value. The higher the measured P value, the greater the
degree of polarization. The term "P.sub.S " is used to refer to the P
value of an aliquot of lymphocytes that has been challenged with a
challenging agent such as an SCM factor of the present invention.
Similarly, the term "P.sub.C " is used to refer to the P value of an
aliquot of lymphocytes not challenged with a challenging agent. When
P.sub.S is compared with P.sub.C, a ratio of P.sub.S to P.sub.C of less
than about 0.9 is an indication of the presence of malignancy in the body
of the donor of the challenged lymphocytes.
A preferred method of using the SCM factor as a challenging agent in the
SCM test comprises comparing P.sub.S to the fluorescence polarization
value, P.sub.M of another aliquot of the lymphocytes contacted with a
mitogen such as phytohaemagglutinin (PHA), to determine an SCM response
ratio, RR.sub.SCM where RR.sub.SCM =P.sub.S .div.P.sub.M. An RR.sub.SCM of
less than about 0.9 indicates the presence of a malignancy. The use of the
RR.sub.SCM is preferable because lymphocytes from donors free of
malignancy respond to PHA but not to cancer-associated SCM factors, while
lymphocytes from donors with malignancy do not respond to PHA but do
respond to cancer-associated SCM factors. This double change in response
pattern gives a sharper indication of the presence of a malignancy.
B. Immunochemical Uses of the SCM Factors
As discussed above, antibodies can be produced against SCM factors by
immunizing antibody-producing animals either with the SCM factors
themselves or SCM factors conjugated to carrier proteins such as keyhole
limpet hemocyanin (KLH). These antibodies can be used for a number of
immunochemical reactions, including assays of SCM factor in body fluids,
detection of cancer cells in biopsies or aspirates by fluorescence
microscopy or flow cytometric methods, and for other purposes discussed
below under "Use of the SCM Factor in the Management of Cancer."
1. Immunoassays for SCM Factor
Once antibodies to SCM factor are produced, either monoclonal or
polyclonal, they can be used in any type of immunoassays including:
competitive or non-competitive sandwich immunoassays; colorimetric assays
(e.g., ELISA, PGLIA (prosthetic-group-label immunoassay), SLIFIA
(substrate-labeled fluorescence immunoassay), etc.); radiometric
procedures such as radioimmunoassay (RIA); and assays employing
luminescence, including both direct and catalyzed chemiluminescence. The
direct chemiluminescence procedures can use luminophores such as
acridinium derivatives; the catalyzed chemiluminescence procedures can use
either enzymic, such as horseradish peroxidase (HRP) or other enzymatic or
non-enzymatic catalysts, including metals. A large number of immunoassays
are known in the art and have been summarized in M. Oellerich,
"Enzyme-Immunoassay: A Review," J. Clin. Chem. Clin. Biochem. 22. 895-904
(1984) and C. Blake & B. J. Gould, "Use of Enzymes in Immunoassay
Techniques," Analyst 109, 533-547 (1984), both of which are incorporated
herein by this reference. For all of these types of immunoassays except
immunoassays dependent on aggregation of antigen-antibody complexes,
monovalent fragments of antibodies, such as Fab or Fab, fragments can
substitute in some applications for entire bivalent antibody molecules.
One particularly useful type of enzyme-linked immunoassay is the
enzyme-linked immunosorbent assay (ELISA assay). An ELISA assay for
detection of SCM factor is described below in Example 21. Briefly, this
assay entails: (1) attachment of SCM factor to a solid phase, typically
plastic; (2) addition of sample to be assayed; (3) incubation of the solid
phase with rabbit anti-SCM factor antibody; (4) incubation with goat
anti-rabbit IgG antibody labeled with the enzyme alkaline phosphatase; (5)
addition of p-nitrophenyl phosphate, a substrate for alkaline phosphatase;
and (6) measurement of absorbance at 405 nm. In this procedure, only the
alkaline phosphatase bound to antibody attached to the solid phase will
yield color; the greater the quantity of SCM factor in the sample, the
lower the absorbance measured at 405 nm. The ELISA test can be used to
detect the level of SCM factor in ultrafiltrates of blood plasmas (Example
23), the presence of SCM factor in purified preparations from serum-free
supernatant cancer cell media (Example 25), and the presence of SCM factor
in cultured human cancer cells (Example 26).
Because of potential cross-reactivity of anti-SCM antibodies with
.alpha.-1-PI because of the sequence homology between them, these
immunodiagnostic tests are preferably carried out on body fluids from
which .alpha.-1-PI molecules have been removed. The .alpha.-1-PI molecules
can be removed by a number of techniques including, but not limited to,
ultrafiltration through filters with nominal molecular weight cutoffs of
1,000 to 3,000 daltons, by passage through chromatographic columns, or by
binding of .alpha.-1-PI to immobilized proteases such as trypsin, with
which .alpha.-1-PI forms a stable complex.
2. Detection of Cancer Cells in Biopsies and Aspirates by Fluorescence
Microscopy or Flow Cytometric Methods
Anti-SCM antibodies labeled with fluorescent markers can be used for
detection of cancer cells producing SCM in biopsies or aspirates by
standard fluorescence microscopic and flow cytometric methods.
C. Detection of SCM-specific Receptors
It is believed that the effects of SCM factor on SCM-responding lymphocytes
are mediated by the specific binding of SCM factor to SCM-factor-specific
receptors located in the cell membrane of the lymphocytes. These receptors
can be detected by the use of labeled SCM molecules, such as radiolabeled
SCM factor, fluorescence-labeled SCM factor, enzyme-labeled SCM factor, or
SCM factor labeled with a chemiluminescent label. Alternatively, SCM
factor can be conjugated to biotin. Avidin or streptavidin can then be
labeled with enzymes, fluorescent labels, or radioactive labels. The
labeled avidin or streptavidin can be used to bind the biotin-conjugated
SCM factor for labeling.
D. DNA Sequences and Vectors
1. Design and Synthesis of Oligonucleotide Sequences
The determination of amino acid sequences for both isolated and purified
SCM factors, as well as the known amino acid sequence of the synthetic SCM
factor, allows the construction of DNA oligonucleotide sequences
corresponding to these amino acid sequences. The construction of these
oligonucleotide sequences varies somewhat depending on whether their
desired use is to be expressed in an in vitro expression system or to
detect the natural gene or genes for SCM factor present in the DNA of the
human genome. However, in either case, the oligonucleotides are
synthesized according to well-known techniques, such as the
phosphotriester method or the phosphite triester method, as described in
K. Itakura, J. J. Rossi, & R. B. Wallace, "Synthesis and Use of Synthetic
Oligonucleotides," Annu. Rev. Biochem. 53, 323 (1984).
a. Sequences for Expression
If the ultimate use of the sequence is to be for the production of
genetically-engineered SCM factor by expression in an in vitro system,
then it is only necessary to synthesize one DNA sequence corresponding to
any particular amino acid sequence. However, the genetic code is
degenerate, and the use of different codons for the same amino acid
affects the rate of translation of the sequence in the host cell. It is
desirable to select codons for those amino acids for which there is a
choice according to the preferred codons for translation in the particular
host organism from which the expression system used is derived. Because
codon usage varies as between bacteria and eukaryotes, it is desirable to
vary the exact sequence of the DNA according to the host in which the DNA
sequence is to be expressed. The differences in codon usage between
bacteria and mammals, including humans, are well-known in the art.
b. Sequences for Detection
If the synthesized DNA sequence is to be used to detect the natural genes
for SCM factor in DNA, different considerations enter into the selection
of the nucleotide sequence. To obtain accurate hybridization, it is
frequently desirable to use a multiplicity of sequences so that all
possible sequences corresponding to the desired amino acid sequence are
present. As one possible example, the region between amino acids 14 and 22
is nearly invariant in the synthesized as well as in the purified SCM
factors, being either F-L-M-I-D-Q-N-T-K or F-L-M-I-E-Q-N-T-K. If all
possible codon combinations were used, however, there would be 2,304
27-nucleotide-long sequences that could correspond to these amino acid
sequences. This number of different sequences is too great to handle
efficiently in a hybridization reaction, so a possible compromise is to
eliminate codons that are the least likely to be used in human DNA,
according to R. Lathe, "Synthetic Oligonucleotide Probes Deduced from
Amino Acid Sequence Data: Theoretical and Practical Considerations," J.
Mol. Biol. 183, 1 (1985). For example, if the three codons least likely to
be used are eliminated for leucine, the single least used codon eliminated
for isoleucine, and the two least used codons eliminated for threonine,
there would be a possible total of 384 separate 27-nucleotide-long
sequences. This number of separate sequences has been used, for example,
in R. B. Wallace, M. J. Johnson, T. Hirose, T. Miyake, E. H. Kawashima,
and K. Itakura, "The Use of Synthetic Oligonucleotides as Hybridization
Probes. II. Hybridization of Oligonucleotides of Mixed Sequence to Rabbit
.beta.-Globin DNA," Nucl. Acids Res. 9, 879-894 (1981).
2. Incorporation of DNA Sequences Into Vectors
Vectors for transfection or transformation contain the DNA sequence coding
for the SCM factor and control sequences such as promoters and enhancers
operatively linked to the coding sequences. The vectors also contain
suitable replication sites. Both the control sequences and the replication
sites are active in the intended host strain. When the host strain is the
bacteria Escherichia coli, plasmids such as pBR322 are suitable vectors.
When the host strain is a cultured mammalian cell such as a human cell,
the vector is typically a virus such as SV40. Suitable host-vector systems
are well-known in the art and are described, for example, in B. Perbal, "A
Practical Guide to Molecular Cloning" (2d ed., John Wiley & Sons, 1988)
and "Guide to Molecular Cloning Techniques" (S. L. Berger and A. R.
Kimmel, eds., Academic Press, 1987; Volume 152 of Methods in Enzymology).
Techniques for incorporation of the desired sequences into the vectors by
ligation, for transfection or transformation of the host cells with the
ligated vectors, for propagation and selection of the host cells
containing the vectors, and for the expression of the SCM factor coded for
by the DNA sequences are all well-known in the art and are described in
the Perbal or Berger & Kimmel references, supra.
IV. RELATIONSHIP BETWEEN THE SCM FACTOR AND CANCER
A. Protection of Cancer Cells in Vivo by SCM Factor
SCM factor molecules are now known to be synthesized by cancer cells. The
SCM factor gene could be the result of genetic damage caused by chemical
or viral agents, or it could be an oncogene whose normal counterpart is
present in the cell. Like other known .alpha.-1-PI variants, the SCM
factor gene or genes are likely derepressed by tumor-inducing agents and
expressed when the mechanisms regulating cell division are activated by
tumor promoters. Proteases and their inhibitors are closely implicated in
the regulation of cell replication, division, maturation, and
fertilization processes. A genetic defect causing production of a
functionally abortive part of the .alpha.-1-PI molecule could contribute
to the erratic and uncontrolled proliferation of initiated and promoted
cancer cells. Our results suggest that SCM factor molecules can protect
cancer cells and enhance their metastatic spread in several ways:
stimulating DNA synthesis of cancer cells (Example 27), protecting the
activity of cancer-cell-associated proteases (Example 28), and
circumventing the immune surveillance and other anti-tumor responses of
the host. One particular protective effect is the suppression of natural
killer (NK) activity of lymphocytes by the SCM factor (Examples 13 and
31). Another possible protective effect is scavenging the reactive
oxygen-containing species released by macrophages against cancer cells via
the methionine residues incorporated in the SCM factor sequence.
Another indication of the common origin of .alpha.-1PI and the SCM factor,
as well as the homology between them, is our discovery that a protein
present in the 50 to 100 kilodalton molecular weight fraction of blood
plasma in donors both having cancer and free of cancer can modify or
abolish the SCM response to the SCM factor in the SCM-responding
subpopulation of lymphocytes from cancer patients. This protein can also
restore the SCM response to PHA in these cells. This SCM response
modifying factor was originally designated as "plasma factor 2" (PF2).
Incubation of SCM-responding lymphocytes from cancer patients for 2.5
hours in the presence of the 50 to 100 kilodalton molecular weight
fraction of plasma from allogeneic or autologous donors removes, on
subsequent washing of the lymphocytes, the receptors for cancer-associated
factors, including SCM factor, and restores the ability of these
lymphocytes to respond again to PHA. That is, the protein reverts the
RR.sub.SCM from values typical of lymphocytes from donors with cancer
(less than 1) to values typical of lymphocytes from donors free of cancer
(greater than 1). PF2 protein, unexpectedly, is identical with
.alpha.-1-PI (Example 29). The .alpha.-1-PI protein can reverse the SCM
response of normal lymphocytes that have been treated with SCM factor, and
can also reverse the SCM response of lymphocytes from donors afflicted
with cancer.
B. Use of the SCM Factor in the Detection and Management of Cancer
As previously detailed in our patent application Ser. No. 07/167,007, the
SCM factor of the present invention can be used for a number of purposes
both in the detection and in the management of cancer.
1. Detection of Cancer
a. Use of SCM Factor as Challenging Agent
SCM factor, or any of its active fragments, can be used as a challenging
agent in the SCM test for the detection of cancer. Lymphocytes from donors
with cancer, but not from donors free of cancer, are primed to respond to
cancer-associated factors in the SCM test. Accordingly, only lymphocytes
from donors with cancer respond to SCM factor with a decrease in
intracellular fluorescein fluorescence polarization value in the SCM test.
This response constitutes an early warning that cancer cells producing SCM
factor are present in the body of the lymphocyte donor, even when the
number of tumor cells or the size of the tumor might not be otherwise
detectable.
b. Detection of Receptors Specific for SCM Factor
SCM factor molecules or fragments that are labeled can be used to detect
the presence of receptors for SCM molecules on the SCM-responding fraction
of lymphocytes. The label can be, but is not limited to, a radioactive
label, a fluorescent label, a chemiluminescent label, or an enzyme label.
The presence of these receptors is itself an indication of cancer. They
can be detected using flow cytometry, fluorescence microscopy,
enzyme-linked assays, or other assays for lymphocyte receptors. If the SCM
molecules are labeled with radioactive isotopes, autoradiography,
scintigraphy, and other detection methods for radionuclides can be used to
detect the presence of receptors for SCM factors.
If SCM-responding lymphocytes are isolated, washed, and incubated with a
saturating quantity of labeled SCM factor, the extent of the binding of
the SCM factor to the lymphocytes indicates the number of SCM factor
receptors present per lymphocyte. This test can be used to indicate the
sensitization of SCM-responding lymphocytes to the SCM factor and can be
used as an alternative to the SCM test to detect the presence of cancer;
it can also be used to confirm the findings of the SCM test.
c. Detection of SCM Factor Molecules in Cancer Biopsies
By flow cytometry, fluorescence microscopy, or enzyme-linked assays, SCM
factor molecules can be detected in cancer biopsies using appropriately
labeled anti-SCM factor antibodies. Because SCM factor molecules are
produced in quantity by cancer cells, their presence in biopsy specimens
is a strong confirmation of the cancerous nature of the tissues from which
the biopsy specimen is taken.
d. Detection of SCM Factor Molecules in Body Fluids
As shown above, SCM factor molecules are excreted by cancer cells into body
fluids such as blood plasma or urine. The presence of SCM factor in body
fluids can therefore be used as a general cancer-specific marker. The
presence of SCM factor molecules can be detected in ultrafiltrates of
cancer patients' blood plasma using antibodies against SCM factor in the
immunoassays described above under "Immunoassays for SCM Factor"; either
polyclonal or monoclonal antibodies of sufficient specificity can be used
in the immunoassays. Antibodies to fragments of the SCM factor can
substitute for antibodies to the entire SCM molecule in many applications.
As the production of SCM factor molecules by cancer cells is decreased by
inhibitors of protein synthesis, the concentration levels of SCM factor in
body fluids can be used to indicate the metabolic activity of any
remaining cancer cells following treatments, and to detect the recurrent
growth of cancer or the presence of otherwise occult metastases. The
presence of SCM molecules could in addition serve as a warning that cancer
cells present in the body of the patient are likely to metastasize.
2. Treatment of Cancer
As detailed above, the SCM factor protects cancer cells against normal
defense mechanisms and promotes their growth and spread. The mechanism by
which the SCM factor accomplishes this includes, but is not limited to,
protection of cancer cells from the action of natural killer (NK) cells
(Examples 13 and 31). This effect of SCM factor leads to the idea that
measures that selectively decrease the in vivo activity of the SCM factor
can be useful in the management of cancer.
As a first step of such methods, once lymphocytes from a known or suspected
cancer patient have been shown to give a positive response in the SCM test
with the SCM factor of the present invention as a challenging agent, a
sample of a body fluid can be taken from the patient, passed through an
ultrafilter with a nominal molecular weight cutoff of 1,000 daltons, and
the fraction passing through the ultrafilter collected and used as an
autologous cancer factor to challenge lymphocytes from the same patient in
the SCM test to confirm the presence of the SCM factor in the fraction. It
need not always be necessary to perform this confirmatory test,
particularly if other clinical indicators indicate the presence of cancer.
a. Inactivation of SCM Factor in Body Fluids
Once the presence of SCM factor in a body fluid of a cancer patient is
shown or inferred, the body fluid can be treated by one of several methods
to reduce the in vivo effect of the factor by inhibiting its production,
selectively removing it, or selectively inactivating it, and the body
fluid can then be returned to the patient, thereby enhancing the
resistance of the patient to the malignancy. Since the SCM factor of the
present invention causes a response in the SCM test regardless of the type
of cancer afflicting the patient, it is believed that reducing the in vivo
effect of the SCM factor can enhance the resistance of the patient not
only to the particular type of cancer originally diagnosed, but also to
any other type of malignancy that might subsequently develop in the
patient. This can prove significant when treating patients with drugs that
have an immunosuppressant effect, or patients with an already compromised
immune system due to conditions such as AIDS.
(1) Inactivation by Dialysis
When the body fluid is peripheral blood, one method of reducing the in vivo
activity of the SCM factor is to physically remove it by dialysis of the
peripheral blood to remove peptides with an apparent molecular weight of
1,000 daltons or less, since the factor will pass through ultrafilters
with a nominal molecular weight cutoff of 1,000 daltons or less, even
though its actual molecular weight is somewhat greater.
(2) Inactivation by Reaction with Antibodies or Antisense Peptides
Another method of reducing the in vivo activity of the SCM factor in a body
fluid is to neutralize it or inactivate it with antibodies specific for
the factor. The antibody is prepared as described above and can be
polyclonal or monoclonal. Alternatively, monovalent antibody fragments,
such as Fab fragments or Fab' fragments, can be used. Use of monovalent
fragments can be preferable to use of intact antibody in some applications
if the formation of large SCM factor-antibody complexes is considered
undesirable. The presence of such large antigen-antibody complexes in the
peripheral blood can possibly cause serum sickness and other allergic
reactions.
As an alternative to the use of antibodies, antisense peptides encoded by
the antisense strand of the DNA whose sense strand encoding for SCM factor
can also be used to inhibit the activity of SCM factor. As reported in Y.
Shai, T. K. Brunck, & I. M. Chaiken, "Antisense Peptide Recognition of
Sense Peptides: Sequence Simplification and Evaluation of Forces
Underlying the Interaction," Biochemistry 28, 8804-8811 (1989), and G.
Fassina, M. Zamai, M. Brigham-Burke, & I. M. Chaiken, "Recognition
Properties of Antisense Peptides to Arga-vasopressin/Bovine Neurophysin II
Biosynthetic Precursor Sequences," Biochemistry 28, 8811-8818 (1989),
peptides that are encoded by the antisense strand of a DNA molecule whose
sense strand encodes a physiologically active peptide often interact
specifically with that peptide. The "sense strand" is the strand of the
DNA identical in sequence with the messenger RNA corresponding to it
(except for the substitution of U in mRNA for T in DNA), while the
"antisense strand" is complementary to the sequence of the mRNA. For
example, if the sense strand has the sequence ATG, the antisense strand
would have the sequence CAT.
As applied to inhibition of SCM factor, the antisense peptide has an amino
acid sequence encoded by the antisense strand of a DNA sequence whose
sense strand encodes a cancer recognition factor of at least 9 amino acid
residues including a core sequence of 9 amino acid residues having an
amphipathicity profile substantially equivalent to the sequence
F-L-M-I-D-Q-N-T-K. Typically, the cancer recognition factor has the amino
acid sequence F-L-M-I-D-Q-N-T-K, corresponding to residues 14-22 of the
synthetic SCM factor molecule, or the sequence
M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K, corresponding
to the entire synthetic SCM molecule.
b. Tagging Anti-SCM Factor Antibody with Anti-cancer Substances
Because SCM factor is produced by actively metabolizing cancer cells
(Examples 25 and 26), an antibody specific for the SCM factor can be used
to target an anti-cancer substance to such cells by labeling anti-SCM
factor antibody with the anti-cancer substance. This directs the
anti-cancer substance to the site of the cancer and thereby raises the
effective concentration of the anti-cancer substance at the site of the
cancer. This procedure can be especially advantageous when the anti-cancer
substance is one that produces side effects when given in larger doses.
Such labeling can be performed by standard conjugation procedures used for
conjugating enzymes, fluorescent labels, or radioactive labels to
antibodies as described in P. Tijssen, "Practice and Theory of Enzyme
Immunoassays," (Elsevier, Amsterdam, 1985), pages 221-278.
c. Use of Non-homologous Protease Inhibitors
Effective control of the proliferation and invasive spread of cancer cells
enhanced by SCM factor might be achieved by inhibition of the proteases
that are protected against natural inhibitors by the SCM factor by using
natural inhibitors that are non-homologous with the SCM factor but of the
same small size and ease of diffusion. Preferably, the inhibitor is
non-homologous with any other protease inhibitor that is substantially
inhibited by SCM factor. Examples include the synthetic variant of
Cucurbita maxima trypsin inhibitor, a 29-residue peptide. Such natural or
synthetic protease inhibitors should be selected for their ability to
overcome the protective effect of the SCM factor. Screening of such
potential protease inhibitors should therefore be carried out on proteases
protected by the SCM factor, either by the entire SCM factor or by the
portion of the molecule active in inhibition of .alpha.-1-PI, the
amino-terminal seven residues. We suggest that the use of such protease
inhibitors non-homologous with .alpha.-1-PI and capable of inhibiting
proteases in the presence of SCM factor could be used for cancer
treatment. The simultaneous removal of SCM factor from blood plasma by
dialysis might help to diminish its effect on the patients' defense
mechanisms.
d. Use of Inhibitors of Protein Synthesis to Inhibit SCM Factor Formation
Because SCM factor is the result of active protein synthesis by cancer
cells, new synthesis of SCM factor can be decreased by treatment with
suitable clinically acceptable non-toxic inhibitors of protein synthesis
that causes a decrease in production of SCM factor by tumor cells, such as
cycloheximide or ascorbic acid. We have previously demonstrated that
ascorbic acid can selectively induce the transition of mitochondria into
the idling, orthodox conformation in cancer cells, thereby decreasing
their metabolic activity. Example 30 shows the effect of ascorbic acid on
the synthesis of SCM factor in MCF7 human breast cancer cells in culture.
Addition of 10.sup.-3 M ascorbate decreased the synthesis of SCM factor in
these cells considerably as measured by the ELISA procedure using anti-SCM
antibodies. The use of ascorbic acid or other protein synthesis inhibitors
is therefore proposed as a cancer-specific, non-toxic inhibitor of SCM
factor synthesis. It could be used on its own or in conjunction with other
methods of removing or inactivating SCM factor.
e. Reversing NK-Suppressive Action of SCM Factor
Because the NK-suppressive activity of SCM factor is believed to protect
cancer cells from natural defenses, one way of restoring the effectiveness
of those defenses is by reversing the NK-suppressive effect of SCM factor.
Such a method can comprise administering to a patient at least one of
whose body fluids contains SCM factor a SCM-factor-inhibiting substance in
a quantity sufficient to substantially reverse the NK-suppressive action
of the SCM factor and substantially restore normal NK activity of
lymphocytes of the patient as measured by in vitro lysis of K562 cells by
the lymphocytes. The SCM-factor-inhibiting substance can be antibodies to
SCM factor, univalent antigen-binding fragments of antibodies to SCM
factor, or antisense peptides whose amino acid sequences are those encoded
by the antisense strand of DNA sequences whose sense strand encodes a
NK-suppressive peptide sequence.
3. Use of Anti-SCM-factor Antibodies to Image Cancer Cells
Because the SCM factor is produced by cancer cells and is found in
association with them, anti-SCM-factor antibodies can also be used to
image cancer cells by labeling the antibodies with imaging substances such
as fluorescent dyes or radioactive isotopes. Such labeled antibodies can
be used to detect cancer cells in biopsies by fluorescence microscopy or
autoradiography. Fluorescent-labeled antibody can also be used for
automated detection of cancer cells by flow cytometry.
4. Use of SCM Factor to Modulate Immune System Activity
Because the SCM factor is capable of inhibiting the NK activity of
lymphocytes (see Examples 13 and 31), it can be used to modulate immune
system activity and assess the effectiveness of anti-cancer drugs. Example
31 shows that the NK-suppressive activity of SCM factor is present only in
the entire synthetic SCM factor molecule (29 amino acids) and fragment F2
(amino acids 8-29 of synthetic SCM factor). Peptides derived from fragment
F2 but incorporating one or more conservative amino acid substitutions are
also expected to have NK-suppressive activity.
Such peptides having NK-suppressive activity can be used in a method for
assessing the effectiveness of an anti-cancer agent capable of inhibiting
the growth of malignant cells in a cell culture. The cell culture includes
both lymphocytes exhibiting NK activity and malignant cells. The method
comprises the steps of: (1) incubating the cell culture with a
substantially purified NK-suppressive peptide in a quantity sufficient to
substantially suppress the NK activity of the lymphocytes of the cell
culture; (2) adding the anti-cancer agent to the cell culture in a
quantity sufficient to inhibit the growth of the malignant cells; and (3)
determining the effect of the anti-cancer agent on the malignant cells by
measuring the inhibition of growth of the malignant cells in the essential
absence of NK activity of the lymphocytes.
Such NK-suppressive peptides can also be used in a method of suppressing
the NK activity of lymphocytes. The method comprises administering to the
lymphocytes a substantially purified NK-suppressive peptide in a quantity
sufficient to substantially suppress the NK activity of the lymphocytes as
measured by a standard test for NK activity. Such a standard test is the
in vitro lysis of K562 cells. This method of suppressing NK activity can
be useful when it is desired to modulate immune system activity. A
clinical example in which such modulation is desirable is the transfusion
of blood into patients who have just undergone a tissue transplant and are
at risk of rejection of the transplant; the lymphocytes of such blood can
be treated with the NK-suppressive peptide to diminish the risk of such
transplant rejection.
Similarly, a substantially purified SCM factor or NK-suppressive peptide
can be used as an immunosuppressive drug. The method of use comprises
administering an immunosuppressive fraction alone or in combination with a
pharmaceutically acceptable carrier in a quantity sufficient to create a
degree of immunosuppression capable of enhancing allograft survival. The
immunosuppressive fraction can be a substantially purified naturally
occurring SCM factor, the 29-amino-acid synthetic SCM factor, or a
NK-suppressive peptide that includes at least residues 8-29 of the
synthetic SCM factor or is derived from that sequence by the occurrence of
conservative amino acid substitutions.
EXAMPLES
The following Examples illustrate: (1) the isolation, purification,
characterization, and activities of substantially purified SCM factor from
body fluids of patients with cancer and (2) the characterization and
activities of synthetic SCM factor and peptides comprising partial
sequences of synthetic SCM factor. These Examples are for illustrative
purposes only and are not to be construed as limiting the invention.
Example 1
Initial Purification of the General Cancer-Associated SCM Factor from Blood
Plasma
Blood samples from patients positively diagnosed as having active cancer,
such as cancer of the breast, lung, colon, ovary, cervix, uterus, larynx,
or skin (basal cell carcinoma and malignant melanoma) were collected into
heparinized vials such as Vacutainer.TM. tubes. Twenty-milliliter portions
of the blood samples were centrifuged at about 1200.times.g for
approximately 40 min. The plasma above the sedimented blood cells was
collected and filtered by pressure through a porous membrane filter such
as an Amicon UM2 or YM2 filter, with a 1000-dalton molecular weight
cutoff. These ultrafiltrates were lyophilized or stored at 4.degree. C.
until further purification.
Example 2
SCM Activity of Initially Purified SCM Factor from Example 1
Aliquots of the ultrafiltrate from each sample of Example 1 were incubated
with potentially SCM-responding lymphocytes obtained from the same donors
and the lymphocytes checked for their SCM response in accordance with the
SCM test procedure described above. In every case the ultrafiltrate caused
the SCM-responding lymphocytes to respond characteristically with a
decrease in P value, as they would have if they had been contacted with
the cancerous tissue itself or with extracts of cancerous tissue (Table
1). As used throughout, "Ca" designates "cancer."
TABLE 1
______________________________________
SCM ACTIVITY OF ULTRAFILTRATES OF EXAMPLE 1
SCM Response:
Diagnosis of Diagnosis of P Value as %
Lymphocyte Donor
SCM Factor Donor
of Control
______________________________________
Malignant Melanoma
Malignant Melanoma
75.7
Malignant Melanoma
Basal Cell 82.0
Carcinoma-Skin
Ca-Larynx Ca-Larynx 62.9
Ca-Breast Ca-Breast 76.0
______________________________________
The data of Table 1 show that even when present in the crude ultrafiltrate,
the SCM factor caused a decrease in the P value of the SCM-responding
lymphocytes from donors afflicted with cancer at least equivalent to the
decrease of the P values observed when lymphocytes are stimulated by crude
extracts of cancerous tissues or cancerous tissues themselves. The
decrease in P value on stimulation by the ultrafiltrates was at least 10%,
which is characteristic of such a positive SCM response. However, the SCM
factor did not pass through the Amicon.TM. UM05 filter with a nominal
500-dalton molecular weight cutoff. These data confirm the small size of
the factor while indicating that the activity is larger than a small
molecule such as a single amino acid.
Example 3
Further Purification of the SCM Factor of Example 1
The lyophilized powder from the samples of Example 1 was dissolved in 2 ml
of sterile preservative-free water for injections. At this stage, the SCM
activity of the preparations was ascertained, and active samples from
donors with the same type and site of cancer were pooled. The pooled
samples were desalted on an 0.9.times.18 cm column of Sephadex.TM. G-10,
which has a fractionation range of from 0 to 700 daltons. The sample
volume per column chromatographic run did not exceed 25% of the column
volume. Elution was carried out with double distilled water at the linear
elution speed of 8 to 9 cm/hr. The desalting was carried out at room
temperature (21.degree.-23.degree. C.). One-ml fractions eluting at
between 0.3 and 0.5 times the total chromatographic bed volume were
collected and the optical densities of the fractions determined. The SCM
activity was contained within the first elution peak. The presence of SCM
activity in that peak was confirmed by an SCM test. An aliquot of the
first elution peak, prepared from an ultrafiltrate originally derived from
plasma of a patient with breast cancer, reduced the P value of lymphocytes
from a patient with breast cancer to 86.3% of the control value in the SCM
test, indicating the presence of SCM activity. These fractions were
collected and lyophilized.
The eluate was further purified by fractionation on a Sephadex.TM. G-50 gel
filtration column, which has a fractionation range of from 1500 to 30,000
daltons. The lyophilized desalted samples were dissolved in 50 mM NH.sub.4
HCO.sub.3, loaded at no more than 5% of the column volume on a
0.9.times.18 cm Sephadex G-50 column at the linear elution speed of 3
cm/hr. The elution was carried out at room temperature, and one-milliliter
fractions eluting from the column at between 0.4 and 0.6 times the total
chromatographic bed volume were collected. These fractions were tested for
SCM activity. Results of these tests are given below in Example 4. The
SCM-active fractions were contained within the first elution peak as
determined by optical densities of the one-milliliter fractions after
testing of the fractions in the SCM test.
Once the fractions were tested for SCM activity, the active fractions from
the same cancer types were pooled and lyophilized.
For further purification the lyophilized samples were dissolved in 10 mM
NH.sub.4 HCO.sub.3 and loaded at no more than 4% of the column volume on
an 0.8.times.26 cm column of Whatman DE-52 microgranular DEAE-cellulose.
The column was washed with 10 ml of 10 mM aqueous NH.sub.4 HCO.sub.3
increasing by 0.108% per minute from 10 mM to 1M NH.sub.4 HCO.sub.3.
One-milliliter fractions were collected and the optical absorption at 220
nm was determined for each fraction. Based on the optical absorbance,
active fractions eluting from the column at between 4.5 and 4.7 times the
total chromatographic bed volume were pooled and lyophilized for testing
and further purification. Results from SCM testing of the active fractions
are given in Example 4.
Example 4
SCM Activity of Further Purified Preparations of Example 3
Table 2 shows the results when aliquots of the Sephadex G-50 fractions from
Example 3 originally from donors with various types of cancer were used to
challenge lymphocytes from donors, also with various types of cancer, in
the SCM test. It can be seen that potentially SCM-responding lymphocytes
have the same characteristic response to the G-50 fractions as they did to
the previously characterized cancer-associated antigens. This desalted
partially purified proteinaceous material exhibits a generally increased
SCM response as compared to the crude ultrafiltrate for which the results
were shown in Table 1. This increased SCM response is shown by decreased P
values.
TABLE 2
______________________________________
SCM ACTIVITY OF SEPHADEX G-50
FRACTIONS OF EXAMPLE 3
SCM Response:
Diagnosis of Diagnosis of P Value as %
Lymphocyte Donor
SCM Factor Donor
of Control
______________________________________
Ca-Lung Ca-Breast 73.0
Ca-Lung Ca-Cervix 69.6
Ca-Lung Ca-Bronchus 73.6
Ca-Larynx Ca-Bronchus 77.6
Ca-Breast Ca-Bronchus 80.2
Ca-Colon Ca-Breast 63.5
Ca-Larynx Ca-Breast 63.0
Malignant Melanoma
Malignant Melanoma
74.9
Healthy Donor
Malignant Melanoma
99.3
Healthy Donor
Ca-Colon 98.0
Colitis Ca-Colon 98.9
______________________________________
Table 3 shows the results when SCM factor obtained from donors with various
types of malignancies after purification through the DEAE-cellulose stage
was used to challenge lymphocytes isolated either from donors with various
types of malignancies or from donors free of malignancy in the SCM test.
As expected, the lymphocytes from cancer patients responded to the SCM
factors purified from the DEAE-cellulose columns with a considerable
decrease in P value, while lymphocytes from donors free of malignant
disease showed no such decrease in P value.
TABLE 3
______________________________________
SCM ACTIVITY OF DEAE-CELLULOSE
FRACTIONS OF EXAMPLE 3
SCM Response:
Diagnosis of Diagnosis of P Value as %
Lymphocyte Donor
SCM Factor Donor
of Control
______________________________________
Ca-Breast Ca-Breast 69.2
Ca-Breast Ca-Bronchus 69.5
Ca-Breast Ca-Cervix 69.0
Basal Cell Basal Cell 82.0
Carcinoma-Skin
Carcinoma-Skin
Healthy Donor
Ca-Colon 98.6
Cholecystitis
Malignant 100 0
Melanoma
Urethritis Ca-Cervix 99.8
Appendicitis Ca-Colon 98.0
Benign Breast
Ca-Breast 97.8
Growth
Benign Pituitary
Ca-Brain 100.0
Adenoma
______________________________________
Example 5
Final Purification of SCM Factor of Example 3 by RP-HPLC
The DE-52 general cancer-associated SCM-active fractions of Example 4 were
then reconstituted and purified to homogeneity by reverse phase high
pressure liquid chromatography (RP-HPLC) using a 2.1 mm.times.22 cm HPLC
column. The column was packed with Aquapore RP-300.TM. (7 microns). The
mobile phases used in the RP-HPLC purification step were as follows:
Phase A: 0.1 volume percent aqueous trifluoroacetic acid (TFA).
Phase B: 0.09 volume percent aqueous TFA in aqueous 70% acetonitrile.
Lyophilized DE-52 SCM-active fractions were reconstituted with sterile
water for injections (without preservatives) and 250 microliter aliquots
were injected into the RP-HPLC column. The mobile phase flow rate was 50
microliters per minute and its composition profile was 10 minutes of 90
volume percent of Phase A, 10 volume percent of Phase B, followed by 30
minutes of linear increase of Phase B at the rate of 3 volume percent per
minute. The optical density peaks detected by optical absorbance at 220 nm
were hand-collected via a "nanobore" teflon tubing into 1.5 ml plastic
conical Eppendorf centrifuge tubes and the solvent was evaporated in a
vacuum centrifuge. In all cases, the general cancer-associated
SCM-recognition factor eluted from the column at 74 volume percent of
Phase B.
Example 6
Alternative RP-HPLC Purification of SCM Factor
Alternatively, the SCM factor can be purified by performing HPLC using a
4.6 mm.times.25 cm HPLC column packed with ULTRASPHERE.RTM. ODS=(5
microns) distributed by Beckman Instruments, Inc. with the DEAE-52
SCM-active fractions of Example 4. The mobile phases used with this column
were as follows:
Phase A: 0.1 volume percent aqueous trifluoroacetic acid (TFA).
Phase B: 0.1 volume percent TFA in aqueous 70% acetonitrile.
The same general procedure was followed with this column as for the
Aquapore column, except that the mobile phase flow rate was 1.00 ml per
minute and its composition profile was 5 minutes of 70 volume percent of
Phase A, 30 volume percent of Phase B, followed by 20 minutes of linear
increase of Phase B at the rate of 3.5 volume percent per minute. The
optical density peaks were detected at 220 nm and were hand-collected into
siliconized glass test tubes and the solvent was evaporated in a vacuum
centrifuge. When this HPLC system was used, in all cases the purification
of general cancer-associated SCM-recognition factor from nineteen
different cancer types, including squamous cell carcinoma of the cervix,
adenocarcinoma of the breast, adenocarcinoma of the bronchus, and
malignant melanoma, always yielded a single optical density peak of
activity, eluting at 56.3 volume percent of Phase B.
Example 7
SCM Activity of RP-HPLC Purified Preparations of Example 6
For SCM activity testing, of the peptides isolated by RP-HPLC on the
ULTRASPHERE.RTM. column in Example 6, the peptides were reconstituted with
sterile water for injections without preservatives. The SCM activity of
SCM-responding lymphocytes after incubation with these samples is shown in
Table 4. This fraction gives the greatest decrease in polarization value
when used to challenge lymphocytes from donors afflicted with cancer. Two
of the three preparations of SCM factor gave a decrease in polarization
value greater than 40%, a larger decrease than seen with any other
fraction tested. The purified factor, as expected, was non-specific with
respect to the type of cancer afflicting the donor of the lymphocytes
used. Also as expected, the purified factor gave no response when used to
challenge lymphocytes from healthy donors.
TABLE 4
______________________________________
SCM ACTIVITY OF RP-HPLC
FRACTIONS OF EXAMPLE 6
SCM Response:
Diagnosis of Diagnosis of P Value as %
Lymphocyte Donor
SCM Factor Donor
of Control
______________________________________
Ca-Breast Ca-Breast 58.9
Ca-Breast Ca-Lung 57.3
Ca-Colon Ca-Breast 55.4
Ca-Breast Ca-Bronchus 68.0
Healthy Donor
Ca-Breast 99.8
Healthy Donor
Ca-Lung 101.0
______________________________________
Example 8
Identification and Isolation of SCM-Active Tryptic Peptides from SCM Factor
Purified from Blood Plasma of Patients with Breast Cancer and Lung Cancer
Tryptic peptides with SCM activity were isolated from the purified SCM
factors isolated from blood plasma of patients with breast cancer or lung
cancer. The cleavage of the purified factors with trypsin and purification
of the active fragments were carried out by the following procedure:
To prevent adsorption loss of the peptide during lyophilization, the SCM
factor was digested with trypsin in the presence of HPLC eluants. Trypsin
digestion was carried out in 0.1M Tris-HCl buffer, pH 8.3, at 37.degree.
C. for 24 hours using 10 percent by weight of trypsin. The digest was
diluted fourfold with 0.1 volume percent aqueous trifluoroacetic acid, and
was injected into an Applied Biosystems 130A microflow HPLC-separation
system. The tryptic fragments were separated using an Aquapore RP-300
column (200 mm.times.2.1 mm). For the elution of the fragments, the mobile
phase solvents were:
Phase A: 0.1 volume percent aqueous trifluoroacetic acid (TFA).
Phase B: 0.09 volume percent TFA in aqueous 70% acetonitrile.
The mobile phase flow rate was 50 .mu.l per minute and the composition
profile was 10 minutes of 96 volume percent Phase A, 4 volume percent
Phase B, followed by a linear elution gradient comprising a 30 min linear
increase in Phase B at a 3 volume percent per minute rate. The SCM-active
tryptic peptide fragment eluted at 69.6 volume percent of Phase B and 30.4
volume percent of Phase A in a total volume of about 30 microliters.
The tryptic peptide cleaved from the SCM factor purified from patients with
lung cancer was tested for SCM activity in Example 10, below, and found to
be fully active. By comparison with the sequences of the entire isolated
SCM factors determined in Example 14, these tryptic peptides were found to
represent amino acids 8-22 of the SCM factor molecule.
Example 9
Use of the Isolated SCM Factor as the Challenging Agent in the SCM Test
Table 5 summarizes the results obtained by using preparations of the
general cancer-associated SCM recognition factor at various stages of
purification from Examples 1, 3, and 6 as the challenging agent in the SCM
test. When lymphocytes from donors afflicted with a number of different
malignancies were used with the factor of the present invention in the SCM
test, a significant response was seen in all cases. This response is given
in Table 5 as a percent of the control polarization value obtained by
performing the SCM measurement on the same lymphocytes unincubated with
the factor. The smaller the value the greater the response to the factor
in the SCM test. Even the crudest preparation of the factor tested, the
ultrafiltrate, give a decrease in polarization value of from 18.0% to
37.1%, and the most highly purified fraction, purified by RP-HPLC, gave a
decrease in polarization value of as great as 44.6%. The factor of the
present invention is specific and only causes a decrease in polarization
value when used to challenge lymphocytes from donors afflicted with
cancer. Even the RP-HPLC purified fraction caused no decrease in
polarization value when used to challenge lymphocytes from healthy donors.
TABLE 5
______________________________________
EXAMPLES OF SCM ACTIVITY AT DIFFERENT
PURIFICATION STAGES (EXAMPLE 9)
Range of Percentage Decrease
In P Value When Lymphocytes
Stage of Purification
From Cancer Patients Used
______________________________________
Ultrafiltrate of Plasma
18.0-37.1
(1000-dalton Cutoff)
Sephadex G-50 Fraction
19.8-37.0
DE-52 Cellulose Fraction
18.0-31.0
RP-HPLC Fraction
32.0-44.6
______________________________________
No fraction showed any activity when tested against lymphocytes from
healthy donors, or from donors with nonmalignant diseases.
Example 10
Activity of the Tryptic Peptide of Example 8 in the SCM Test
The tryptic peptide obtained from SCM factor from plasma of patients with
lung cancer, whose purification was described above in Example 8, was
fully active in the standard SCM test.
Approximately 5.times.10.sup.-2 femtograms of this fragment (i.e.,
5.times.10.sup.-17 grams, or approximately 16,000 molecules of the
fragment) gave full activity in the test. When the fragment was isolated
from patients with lung cancer, it proved active in the SCM test when
tested against lymphocytes from a patient with small cell lung carcinoma,
and crossreacted fully with lymphocytes from a patient with adenocarcinoma
of the breast, as shown in Table 6. However, no response was seen when
lymphocytes from healthy donors were used.
TABLE 6
______________________________________
SCM ACTIVITY OF TRYPTIC FRAGMENT
OF EXAMPLE 8 FROM LUNG CANCER
SCM Response:
P Value as Percent
Diagnosis of Lymphocyte Donor
of Control
______________________________________
Small Cell Lung Carcinoma
70.0 68.0
Adenocarcinoma of Breast
67.5 68.0
Healthy Donors 102.0 104.0
______________________________________
Example 11
Cross-Reactivity of Isolated SCM Factors
The following example demonstrates the ability of the isolated SCM factor
to cause a response in the SCM test when used to challenge lymphocytes
derived from donors afflicted with dissimilar types of cancer. In order to
demonstrate this cross-reactivity, two milliliters of cell-free blood
plasma was obtained from each of a number of blood samples from cancer
patients. The blood samples had originally been collected into heparinized
Vacutainer.TM. tubes. The samples were ultrafiltered through an Amicon.TM.
UM2 or YM2 filter with a nominal molecular weight cutoff of 1000 daltons
for at least 12 hr and stored under sterile conditions at 4.degree. C..
Potentially SCM-responding lymphocytes were isolated from heparinized
blood samples from patients with cancer, healthy donors, and donors with
non-malignant diseases. To test the activity and cancer specificity of the
general cancer-associated SCM-recognition factor containing
ultrafiltrates, 0.75 ml aliquots of potentially SCM-responding lymphocytes
(5.times.10.sup.6 cells/ml in PBS) were incubated for 40 min at 37.degree.
C. with the ultrafiltrates. 0.075 ml of the ultrafiltrates was used for
each assay. SCM measurements were carried out as previously described in
the article by L. Cercek and B. Cercek, "Application of the Phenomenon of
Changes in the Structuredness of Cytoplasmic Matrix (SCM) in the Diagnosis
of Malignant Disorders: a Review," Europ. J. Cancer 13, 903-915 (1977) and
in the prior patent application by B. Cercek and L. Cercek, Ser. No.
867,079, filed May 27, 1986, and entitled "Method for Measuring Polarized
Fluorescence Emissions." In the SCM assay, a decrease in the intracellular
fluorescence polarization value (P value) of at least 10% was taken as a
positive response to the challenging ultrafiltrate.
TABLE 7
__________________________________________________________________________
CROSSREACTIVITY OF SCM ACTIVITY OF ULTRAFILTRATES
OF EXAMPLE 11
Cases Responding/Cases Tested for Lymphocyte Donor with
Diagnosis of:
Diagnosis of Donor
Ca- Ca- Ca- Ca- Ca-
Ca- Ca- Ca- Non-Cancer
Healthy
of Ultrafiltrate
Mouth
Larynx
Cervix
Ovary
Lung
Brain
Breast
Colon
Diseases
Donors
__________________________________________________________________________
Ca-Lung -- 2/2 3/3 -- 5/5
3/3 3/3 2/2 0/3 0/3
Ca-Bronchus
1/1 1/1 2/2 2/2 2/2
-- 2/2 1/1 -- 0/2
Ca-Breast 1/1 1/1 3/3 2/2 2/2
-- 3/3 1/1 0/3 0/2
Ca-Ovary 1/1 2/2 2/2 3/3 2/2
-- 2/2 -- 0/4 0/2
Ca-Cervix 1/1 1/1 5/5 1/1 2/2
-- 2/2 2/2 0/3 0/3
Ca-Colon 2/2 -- 3/3 -- 2/2
-- 2/2 1/1 0/2 0/2
Ca-Mouth 2/2 -- 1/1 -- 1/1
-- 1/1 1/1 -- 0/2
Ca-Larynx 1/1 6/6 3/3 -- 2/2
-- 2/2 1/1 0/2 --
Malignant Melanoma
-- -- -- -- -- -- 1/1 1/1 -- 0/2
Glioblastoma
-- -- 2/2 -- -- 2/2 2/2 1/1 0/2 0/2
Appendicitis
-- -- -- -- 0/1
-- 0/1 0/3 0/3 0/2
Urethritis -- -- 0/2 -- 0/2
-- 0/1 0/2 0/3 0/3
Infectious Abscess
-- 0/1 0/1 -- -- -- 0/1 0/1 0/2 0/2
Colitis -- -- 0/1 -- -- -- 0/1 0/3 0/2 0/2
Benign Pituitary
-- -- -- -- 0/2
0/2 0/2 -- 0/1 0/1
Adenoma
Healthy Donors
-- -- 0/2 0/1 0/3
-- 0/3 0/1 0/4 0/6
__________________________________________________________________________
As the data of Table 7 show, potentially SCM-responding lymphocytes from
patients with eight different cancer types responded to ultrafiltrates
containing general cancer-associated SCM-recognition factors from nine
different cancer types. In contrast, ultrafiltrates from plasmas of
healthy donors and donors with non-cancer diseases did not trigger any
positive SCM responses. Also, neither did the potentially SCM-responding
lymphocytes from healthy donors or those with non-malignant conditions
respond to any of the ultrafiltrates in the SCM test.
Example 12
Modification of the SCM Response by the Isolated SCM Factor
To demonstrate the modification of the SCM response of lymphocytes free of
malignancy by incubation with the isolated SCM factor, potentially
SCM-responding lymphocytes were isolated from the blood samples of healthy
donors and suspended in complete Dulbecco's phosphate buffered saline
(PBS) at 5.times.10.sup.5 cells/ml as described in the European Journal of
Cancer article, supra, and also in the prior patent application by B.
Cercek, Ser. No. 838,264, filed Mar. 10, 1986, and entitled "Automated
Collection of Buoyant Density Specific Cells from Density Gradients."
Aliquots of these cells were incubated in 3 ml of the following: (a)
cell-free blood plasma from cancer patients; (b) plasma from cancer
patients ultrafiltered for 12 hours through an Amicon.TM. UM2 filter with
a molecular weight cutoff of 1000 daltons; (c) plasma from cancer patients
ultrafiltered for 12 hours through an Amicon.TM. UM5 filter with a nominal
molecular weight cutoff of 500 daltons; (d) general cancer-associated SCM
factor as purified through the desalting or Sephadex G-10 column stage;
(e) the factor as purified through the Sephadex G-50 column stage; (f) the
factor as purified through the DEAE-cellulose column step; (g) the factor
as finally purified through the RP-HPLC step; and (h) plasma from healthy
donors ultrafiltered through an Amicon.TM. UM2 filter with a nominal
molecular weight cutoff of 1000 daltons. These incubations were performed
for 2.5 hr.
The ability of the SCM factor to modify the SCM response of the lymphocytes
from healthy donors was demonstrated by determining the SCM response ratio
(RR.sub.SCM) of the lymphocytes before and after the incubation with each
of the fractions described above. Before being contacted with either a
mitogen or with the SCM factor for determination of the RR.sub.SCM the
incubated cells were thoroughly washed. The presence or absence of
modification was determined by the ratio of the polarization value of the
lymphocyte suspension after a short contact period with substrates
containing the SCM factor over the polarization value of the lymphocyte
suspension after a short contact period with phytohaemagglutinin (PHA). In
accordance with the SCM test procedure, a RR.sub.SCM of less than 1.0 is a
positive indication of the presence of malignancy in the donor while an
RR.sub.SCM of 1.1 or greater indicates the absence of malignancy.
TABLE 8
______________________________________
MODULATION OF SCM RESPONSES OF
LYMPHOCYTES FROM HEALTHY DONORS
BY SCM FACTOR (EXAMPLE 12)
RR.sub.SCM:
SCM Preparation Used
Before After
For Modulation Modulation Modulation
______________________________________
Cell-free Cancer Blood Plasma
1.38 0.80
Ultrafiltrate of Cancer Blood
1.35 0.78
Plasma (1000 Daltons Cutoff)
Sephadex G-10 (SCM-Active)
1.40 0.80
Sephadex G-50 (SCM-Active)
1.38 0.73
DEAE-Cellulose (SCM-Active)
1.39 0.70
RP-HPLC (SCM-Active)
1.40 0.65
Ultrafiltrate of Cancer Blood
1.38 1.38
Plasma (500 Daltons Cutoff)
Ultrafiltrate from Autologous
1.38 1.38
and Allogenic Blood Plasma
from Healthy Donors
______________________________________
Table 8 indicates the effect of the incubation with SCM-factor-containing
fractions on the response of the lymphocytes to either SCM factor or
phytohaemagglutinin, as reflected in the RR.sub.SCM. Lymphocytes which
were either not preincubated, or which were preincubated with
ultrafiltrate from healthy donors filtered through a filter with a nominal
1000-dalton molecular weight cutoff, or which were preincubated with
ultrafiltrate from donors with cancer filtered through a filter with a
nominal 500-dalton molecular weight cutoff, showed an RR.sub.SCM of 1.35
or higher, as expected. By contrast, lymphocytes which were preincubated
With fractions containing SCM factor all showed decreases in the
RR.sub.SCM to a value of 0.65-0.80 characteristic of lymphocytes
originally isolated from patients with malignant disease.
Example 13
Effect of the Isolated SCM Factor on Lymphocyte Cytotoxicity
To demonstrate the effect of the isolated SCM factor on the natural
cytotoxicity of potentially SCM-responding lymphocytes toward malignant
cells, such lymphocytes obtained from healthy donors were incubated for
21/2 hr at 37.degree. C. with plasma containing SCM factor isolated as
previously described from blood samples of donors afflicted with cancer.
Aliquots of these lymphocytes were also retained as controls and not
incubated. In addition, potentially SCM-responding lymphocytes were
obtained from donors having cancer, and treated in the same manner--some
aliquots incubated with plasma containing SCM factor and others retained
as controls and not incubated.
After incubation the cytotoxicity of the lymphocytes was tested in
accordance with the method described in M. R. Potter and M. Moore,
"Natural Cytotoxic Reactivity of Human Lymphocyte Subpopulations,"
Immunology 37, 187-194 (1979). In accordance with this published method
cells of the K 562 human myeloid cell line labeled with .sup.51 Cr were
used as target cells for the assay. The potentially SCM-responding
lymphocytes were used as the effector cells. The ratio of target cells to
effector cells was 1 to 20. Release of the .sup.51 Cr indicates that the
effector cells are toxic to the target cell. The percent of cytotoxicity
is determined as follows:
##EQU3##
where R.sub.S is the percent of .sup.51 Cr release in the sample, R.sub.C
the percent of .sup.51 Cr release in the control and R.sub.T is the
percent of .sup.51 Cr release in the presence of a detergent, Triton
X-100. The results are shown in Table 9. These results show that
incubation of potentially SCM-responding lymphocytes from healthy donors
for 2.5 hr with ultrafiltrates filtered through filter with a nominal
1000-dalton molecular weight cutoff decreased their cytotoxicity by over
90%. When the incubation was performed with potentially SCM-responding
lymphocytes from cancer patients, the decrease in cytotoxicity was
smaller, between 40 and 90%. However, such lymphocytes from cancer
patients had lower levels of cytotoxicity before incubation, and the
residual level of cytotoxicity remaining after incubation with
ultrafiltrate was comparable to that remaining after incubation of
lymphocytes from healthy donors. The lower level of cytotoxicity present
in cells from cancer patients was consistent with a decrease of such
cytotoxicity caused by in vivo exposure to factors such as the
cancer-associated SCM recognition factor.
TABLE 9
__________________________________________________________________________
EFFECT OF SCM FACTOR ON NATURAL LYMPHOCYTE
TOXICITY AGAINST K 562 HUMAN MYELOID CELL LINE
(EXAMPLE 13)
Diagnosis of Donor % Cytotoxicity
of Potentially
Diagnosis of Donor
of Lymphocytes:
SCM-Responding
of SCM Factor as
Before
After % Decrease
Lymphocytes
Ultrafiltrate
Incubation
Incubation
in Cytotoxicity
__________________________________________________________________________
Healthy Donor #1
Ca-Cervix 40.0 2.2 94.5
Healthy Donor #2
Ca-Brochus
30.0 1.7 94.3
Healthy Donor #3
Ca-Larynx 11.0 0.76 93.1
Healthy Donor #4
Ca-Layrnx 22.0 2.2 90.0
Healthy Donor #5
Ca-Pharynx
41.0 0.33 99.2
Ca-Tongue Ca-Larynx 23.0 2.1 90.9
Ca-Lip Ca-Bronchus
7.4 2.7 63.5
Ca-Ovary Ca-Bronchus
10.0 6.1 39.0
Ca-Cervix Ca-Cervix 25.2 1.5 94.0
Ca-Bronchus
Ca-Cervix 29.6 3.1 89.5
Ca-Bronchus
Ca-Cervix 29.6 3.1 89.5
__________________________________________________________________________
Example 14
Amino Acid Sequences of Isolated SCM Factors
The amino acid sequences of isolated SCM factors, determined from purified
preparations from blood plasmas of 12 different cancers, are presented in
Table 10. The sequences were determined by an automated Edman degradation
procedure, using the Applied Biosystems 477A protein sequencer coupled
with an online 120A PTH-amino acid analyzer. Sequence-calling software was
used to establish the amino acid residue at each cycle. The sequences of
the SCM-factor peptides were obtained in repetitive analyses of two to
three different preparations, isolated and purified to homogeneity, from
pooled blood plasmas of about 5 to 50 different patients with a diagnosis
of the same type of cancer. Amino acid residues designated in parentheses
below the primary, most significant residue detected at the particular
degradation cycle represent secondary amino acid residues present in some
of the degradation cycles in significant amounts. These secondary residues
may indicate the presence of genetic polymorphisms of the SCM factors from
individual blood donors contained in the sample pool that was used for
sequencing; many, but not all, of the substitutions in these polymorphisms
are conservative substitutions. In two cases, where a total of 35 amino
acids were seen, the last six were weak. This indicates that two separate
factors were present in the preparations, one of 29 amino acids, and a
second of up to 35 amino acids. These two preparations were from donors
with cancer of the prostate and seminoma of the testes. In some cases, no
amino acid was seen in a particular cycle, designated by "X." These amino
acids are most likely cysteine, and are otherwise referred to as cysteine
(C). This is because of the 20 common amino acids, cysteine is the only
one not detectable by the Edman degradation procedure.
TABLE 10
__________________________________________________________________________
AMINO ACID SEQUENCES OF PURIFIED ISOLATED SCM FACTORS
__________________________________________________________________________
Ca-BREAST:
##STR1##
Ca-LUNG:
##STR2##
Ca-COLON:
##STR3##
MELANOMA:
M I P P E V K F N K P F V F L M I D Q N T K XPX F M G X
SCC-CERVIX:
##STR4##
Ca-OVARY:
##STR5##
Ca-UTERUS:
##STR6##
Ca-PANCREAS:
VI P P E V K F N K P F V F L M I D Q N T K XPLF M G K
Ca-RENAL:
VI P P E V K F N XP F V F L M I D Q N T K V PLF M G K
##STR7##
##STR8##
Ca-PROSTATE:
##STR9##
##STR10##
##STR11##
__________________________________________________________________________
Example 15
SCM Activity of Synthetic SCM Factor
A synthetic SCM factor, representing the "consensus sequence" of
M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-L-I-D-Q-N-T-K-V-P-L-F-M-G-K, was synthesized
using conventional solid-phase peptide synthesis techniques. Such
techniques are described, for example, in M. Bodanszky, "Peptide
Chemistry" (Springer-Verlag, Berlin, 1988), Ch. 10, "Solid Phase Peptide
Synthesis."
The SCM activity of this synthetic SCM factor was tested by the standard
SCM test. The fraction of SCM-responding lymphocytes from patients with a
number of types of cancer and from normal healthy donors was challenged
with the synthetic SCM factor. The factor was dissolved in sterile water
for injections and was administered to the SCM-responding lymphocytes at
190 picomoles per 3.times.10.sup.6 lymphocytes. As can be seen in Table
11, lymphocytes from patients with several types of cancer responded with
significant decreases in intracellular fluorescein fluorescence
polarization to the synthetic SCM factor. Most of these decreases in
fluorescence polarization exceeded 40% and were comparable to the
decreases seen with the most highly purified preparations of SCM factor
isolated from blood plasma. This response was also specific for
lymphocytes from patients with cancer. When lymphocytes from healthy
donors were challenged with synthetic SCM factor, no decrease in
fluorescence polarization was seen, even when the cells were challenged
with increased quantities of SCM factor as high as 960 picomoles per
3.times.10.sup.6 lymphocytes.
Example 16
Fragments of Synthetic SCM Factor
Peptides representing distinct fragments of the synthetic SCM factor of
Example 15 were synthesized by conventional solid-phase peptide synthesis
techniques. These peptides were designated F1-F5 and have the following
sequences:
F1: M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-D-Q-N-T-K;
F2: F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K;
F3: F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K;
F4: F-L-M-I-D-Q-N-T-K; and
F5: M-I-P-P-E-V-K-F-N-K-P-F-V-F.
These fragments represented the following portions of the complete
synthetic SCM molecule: Fl, amino acids 1-22; F2, amino acids 8-29; F3,
amino acids 8-22; F4, amino acids 14-22; and F5, amino acids 1-13.
Example 17
Activity of Fragments of Synthetic SCM Factor of Example 16 in the SCM Test
Fragments F1 through F5, representing different portions of the synthetic
SCM molecule, were used as the challenging agents for both lymphocytes
from patients with cancer and lymphocytes from normal donors in SCM tests.
The SCM tests were performed as described in Example 15. The results are
shown in Table 11. Fragments F1, F2, F3, and F4 were all fully active in
the SCM test, while fragment F5 was inactive. For fragments F1 through F4,
the expected specificity of the SCM response was maintained, as these
fragments gave no decrease in fluorescence polarization when used to
challenge lymphocytes isolated from donors free of malignancy.
Of these peptides representing active fragments of the synthetic SCM
molecule, the smallest is F4, residues 14-22. All of the other active
peptides include this segment, while F5, which does, not have this
segment, is inactive. Accordingly, and based upon these results, residues
14-22 can be considered to be the active site of the synthetic SCM-factor
molecule. Significantly, this region of the peptide is virtually invariant
in the isolated SCM factors, except for the extremely conservative
substitution of glutamic acid (E) for aspartic acid (D) at position 8 in
two of the factors.
TABLE 11
______________________________________
SCM ACTIVITY OF SYNTHETIC SCM
FACTOR AND FRAGMENTS F1 TO F5
SCM Response as % of Control P Value to:
Diagnosis of
Synthetic
Lymphocyte
SCM
Donor Factor F1 F2 F3 F4 F5
______________________________________
Melanoma 58.0 62.8 63.6
61.5 -- --
Ca-Ovary 58.4 -- 65.0
-- 60.2 --
Ca-Breast -- 59.0 -- 61.2 -- --
Melanoma -- 66.4 65.0
59.4 -- --
Ca-Breast 56.7 65.7 -- 63.2 -- --
Ca-Breast -- 68.4 61.5
56.7 -- --
Ca-Liver -- 58.0 58.8
55.0 -- --
Ca-Stomach
60.0 66.7 67.0
-- -- --
Ca-Testis 57.3 -- -- 67.0 56.2 --
Ca-Lung 59.7 -- -- -- 57.0 99.5
Ca-Lung 69.0 73.0 -- 68.5 -- --
Ca-Breast 57.0 -- -- -- 58.0 99.0
Healthy (M,30)
102.0 104.0 -- -- 100.0 --
Healthy (F,29)
100.0 101.0 -- 104.0 103.0 --
Healthy (M,36)
100.5 -- -- 100.0 102.0 --
Healthy (F,59)
99.1 100.0 -- -- 99.2 --
Healthy (M,28)
98.8 100.3 100.5
-- 99.3 --
______________________________________
Concentrations of synthetic SCM factor and fragments F1 to F5 used in
these examples were 190 picomoles per 2 .times. 10.sup.6 lymphocytes.
Fragments represent the following amino acid residues of synthtic SCM
factor: F1 (1-22); F2 (8-29); F3 (8-22); F4 (14-22); and F5 (1-13).
P value denotes intracellular fluorescein fluorescence polarization value
as measured with the SCH test.
Example 18
Amphipathicity Profiles of SCM-active Peptides and Peptide Fragments
FIG. 1 shows the amphipathicity profile of the F4 peptide fragment. For
comparison, FIG. 2 also shows the amphipathicity profile of the synthetic
SCM-active octapeptide whose sequence and SCM activity are disclosed in
our co-pending patent application Ser. No. 07/163,250, filed Mar. 2, 1988,
entitled "Synthetic SCM-Active Cancer Recognition Peptides," and
incorporated herein by this reference. The amphipathicity profiles of the
SCM-active octapeptide and of F4 were nearly identical, even though only 4
of 8 of the amino acids of the octapeptide are homologous with those of
F4.
Table 12 shows hydrophilicity values of the individual amino acids in the
sequence of F4, the synthetic SCM-active octapeptide of our co-pending
patent application Ser. No. 07/163,250, and of the purified experimental
allergic encephalitogenic (EAE) nanopeptide, which is inactive in the SCM
test. The only difference between the synthetic SCM-active octapeptide and
the inactive EAE nanopeptide is an additional serine residue in position
2. Serine has a positive hydrophilicity value (+0.3), while in the active
F4 fragment the first four residues all have negative hydrophilicity
values and in the synthetic SCM-active octapeptide, residues 1, 2, and 4
have negative hydrophilicity values and residue 3, glycine, has a
hydrophilicity value of 0.0. The hydrophilic serine in position 3 of EAE
disrupts the sequence of negative hydrophilicities. This disruption
appears sufficient to prevent the recognition of EAE peptide by the
receptors in the lymphocytes of cancer patients that recognize the
SCM-active peptide. Accordingly, pure EAE peptide purified away from the
SCM-active octapeptide is completely devoid of SCM activity. This example
suggests the importance of amphipathicity profiles in controlling the
recognition of the SCM factor by its corresponding receptor.
TABLE 12
______________________________________
HYDROPHILICITY VALUES (HV) OF AMINO
ACID (AA) SEQUENCES OF SCM-ACTIVE
AND SCM-INACTIVE PEPTIDES
SCM-Inactive
SCM-Active Peptides: EAE-Nano-
F4 Nanopeptide Octapeptide peptide
AA HV AA HV AA HV
______________________________________
F -2.5 F -2.5 F -2.5
L -1.8 W -3.4 S +0.3
M -1.3 G 0.0 W -3.4
I -1.8 A -0.5 G 0.0
D +3.0 E +3.0 A +3.0
Q +0.2 G 0.0 E +3.3
N +0.2 Q +0.2 G 0.0
T -0.4 R +3.0 Q +0.2
K +3.0 R +3.0
______________________________________
Example 19
Induction of SCM Response Characteristic of Malignancy in Lymphocytes from
Healthy Donors by Synthetic SCM Factor
The synthetic SCM factor can induce an SCM response characteristic of
malignancy in lymphocytes from healthy donors. The induction of this
response requires active protein synthesis.
To demonstrate this induction, SCM-responding lymphocytes from normal,
healthy donors were isolated and divided into four aliquots containing
5.times.10.sup.6 cells/ml. Lymphocytes in the first aliquot were suspended
in PBS and were used as untreated controls. Lymphocytes in the second
aliquot were incubated with 400 picomoles of synthetic SCM factor, or,
alternatively, with purified SCM factor from plasma from patients with
cancer. Lymphocytes in the third aliquot were incubated with 400 picomoles
of synthetic SCM factor plus 10 .mu.g/ml of cycloheximide. Lymphocytes in
the fourth aliquot were incubated with 400 picomoles of synthetic SCM
factor plus 10 .mu.g/ml of actinomycin D. All four aliquots were incubated
at 37.degree. C. for 2.5 hours. The aliquots were then washed three times
with PBS, collected by centrifugation, and resuspended in PBS. The
aliquots were then challenged with 190 picomoles of synthetic SCM factor
per 2.times.10.sup.6 cells for 40 min at 37.degree. C. and the
intracellular fluorescein fluorescence polarization was determined for
each sample as previously described. As can be seen in Table 13, untreated
control lymphocytes from a healthy donor did not respond to synthetic SCM
factor in the SCM test; i.e., there was no decrease in fluorescence
polarization in comparison to unchallenged control lymphocytes. In
contrast, lymphocytes primed or induced by pretreatment with synthetic SCM
factor responded with a significant decrease in fluorescence polarization
when challenged either with synthetic SCM factor or with purified SCM
factor isolated from blood plasma of patients with colon cancer. This
response was prevented when the pre-incubation occurred in the presence of
the protein synthesis inhibitors cycloheximide or actinomycin D, thus
indicating that active protein synthesis was need to induce the response
to SCM-active peptides.
TABLE 13
______________________________________
IN VITRO INDUCTION OF SCM RESPONSES IN
NORMAL, HEALTHY LYMPHOCYTES (NHL) BY
PRETREATMENT WITH SYNTHETIC SCM FACTOR
SCM Res-
Treatment of NHL Source of ponse:
Before Challenge Challenging
P Value as
with SCM Factor SCM Factor % of Control
______________________________________
Untreated NHL Control
-- 100.0
NHL + Synthetic SCM Factor
Synthetic 63.5
(2.5 hr incubation); Washed
NHL + Synthetic SCM Factor
Plasma 62.2
(2.5 hr incubation); Washed
(Ca-Colon)
NHL + Synthetic SCM Factor +
Synthetic 104.0
10 g/ml Cycloheximide
(2.5 hr incubation); Washed
NHL + Synthetic SCM Factor +
Synthetic 99.7
10 g/ml Actinomycin D
(2.5 hr incubation); Washed
______________________________________
Example 20
Preparation of Antibodies to Synthetic SCM Factor
The synthetic SCM factor molecule was used to immunize experimental
animals. Both pure synthetic SCM-factor molecules and SCM conjugated to
the carrier keyhole limpet hemocyanin (KLH) via an added carboxy-terminal
cysteine using N-succinyl bromoacetate as the cross-linking agent. These
immunogens were used to immunize female New Zealand rabbits. Both
immunogens were diluted for primary immunization to 1.0 mg/ml with sterile
PBS, combined with an equal volume of Freund's complete adjuvant, and
emulsified. For primary immunization, a total of 25 .mu.g or 50 .mu.g of
either synthetic SCM factor or synthetic SCM factor conjugated with KLH
(SCM-KLH) was injected into each rabbit; two rabbits were used for each
dose range. The inoculate was administered at 0.2 ml into two legs
intramuscularly and over a minimum of 12 dorsal sites subcutaneously at
0.2 ml per site. One month later, the first booster injection was
administered. Synthetic SCM factor and SCM-KLH were each administered with
an equal-volume mixture of Freund's complete and incomplete adjuvants and
emulsified. The booster inoculates were injected via intramuscular and
subcutaneous sites similar to those used for primary inoculations. Total
doses of 25 .mu.g or 50 .mu.g of immunogen per rabbit were administered in
the booster injections.
Blood samples taken 10 weeks after primary immunization yielded antisera
containing higher amounts of immunoglobulins (IgG) from those animals
injected with 50 .mu.g of immunogen than from those animals injected with
25 .mu.g of immunogen. Radial immunodiffusion tests, conducted as
described in W. Becker, "Determination of Antisera Titres Using the Single
Radial Immunodiffusion Method," Immunochemistry 6, 539 (1969), gave
precipitation reactions against the unconjugated SCM factor and SCM factor
conjugated to bovine serum albumin (BSA).
To separate the immunoglobulins containing the desired antibodies from the
antisera, the immunoglobulins were first precipitated with an equal volume
of saturated ammonium sulphate. The precipitates were then dissolved in
0.9% NaCl. To remove ammonium sulphate, the antibody-containing solutions
were either dialyzed or ultrafiltered 10 times through an Amicon.TM.
membrane filter with a 5000-dalton molecular weight cutoff. Antibodies
were kept frozen at -40.degree. C. until use.
Example 21
ELISA Assay for SCM Factor
A double-antibody enzyme-linked competitive immunosorbent assay (ELISA) was
developed for detection of SCM factor by the use of antibodies raised
against SCM factor (Example 20). The ELISA assay is depicted schematically
in FIG. 3. In the first step, SCM factor is attached to a solid phase such
as plastic, typically by passive adsorption. In the second step, the
sample to be assayed, along with a limited quantity of the anti-SCM
antibody, is added. After a thorough washing, an excess of the labeled
second antibody, goat anti-rabbit IgG labeled with the enzyme alkaline
phosphatase, is then added in the third step. The substrate for alkaline
phosphatase, p-nitrophenylphosphate, is then added, and the absorbance at
405 nm (A.sub.405) is measured. In this assay, any free SCM factor added
at the second step competes with the SCM factor adsorbed to the solid
phase. Only the solid-phase SCM to which the first and second antibodies
are bound yields color. Therefore, the higher is the concentration of SCM
factor in the test sample, the lower is the measured A.sub.405. This is
typical of a competitive assay.
Variations on this procedure have been employed to detect SCM molecules in
cancer cells, supernatants of growth culture media, blood plasma
preparations from cancer patients, and purified extracts of SCM from
various sources.
Example 22
Activity of Anti-SCM Antibodies
The activity of the antibodies of Example 20 raised against both
unconjugated SCM factor and the KLH-SCM factor conjugate was determined by
a variation of the ELISA assay of Example 21. Different dilutions of the
antibodies were used, and no sample representing free SCM was added to the
assay. The results are shown in FIG. 4 for the antiserum raised against
unconjugated SCM factor, and in FIG. 5 for the KLH-SCM factor conjugate.
As can be seen, both antibody preparations were active against purified
SCM factor.
Example 23
Determination of SCM-Factor Levels in Ultrafiltrates of Blood Plasmas by
ELISA Assay
The level of SCM factor was determined in a number of ultrafiltrates of
blood plasmas from both healthy donors and cancer patients. Ultrafiltrates
of blood plasmas from 12 cancer patients and 12 normal, healthy donors
were prepared by filtration through an Amicon.TM. YM2 membrane filter with
a 1000-dalton molecular weight cutoff. The level of SCM factor was assayed
immunochemically by the ELISA assay of Example 21. The results are shown
in Table 14. The levels of SCM factor detected by the ELISA assay were in
the nanogram range per milliliter of ultrafiltrate. In the ultrafiltrates
from donors with cancer, they were from 4.8 to 25.5 ng/ml. In normal,
healthy donors, the levels of SCM factor were either below the minimum
detectable level or up to a maximum of 1.85 ng/ml.
TABLE 14
______________________________________
LEVELS OF SCM FACTOR IN AMICON .TM. YM2 ULTRA-
FILTRATES OF BLOOD PLASMAS FROM CANCER
PATIENTS AND NORMAL, HEALTHY DONORS AS
DETECTED BY ANTI-SCM FACTOR ANTIBODIES
IN COMPETITIVE ELISA ASSAYS
Diagnosis of Donor's SCM Factor
Blood Donor Sex and Age
ng/ml
______________________________________
Ca-Breast F 39 12.0
Ca-Breast F 50 10.1
Ca-Breast F 49 7.0
Ca-Lung F 76 13.4
Ca-Lung F 67 8.7
Ca-Lung M 47 5.5
Ca-Pancreas F 50 8.5
Ca-Colon M 42 4.8
Ca-Colon M 44 14.0
Ca-Colon F 60 10.5
Malignant Melanoma
F 38 15.7
Malignant Melanoma
F 50 25.5
Normal Healthy M 31 .sup. ND.sup.a
Normal Healthy M 49 ND
Normal Healthy M 26 0.60
Normal Healthy M 38 1.85
Normal Healthy M 29 1.03
Normal Healthy M 36 1.65
Normal Healthy F 27 0.22
Normal Healthy F 32 0.82
Normal Healthy M 34 ND
Normal Healthy M 46 0.22
______________________________________
.sup.a ND = none detected
Example 24
NH.sub.2 -terminal Amino Acid Sequences of SCM Factor Secreted from Human
Cancer Cells in Culture
The partial NH.sub.2 -terminal amino acid sequences of SCM-factor molecules
present in the supernatant of serum-free culture media in which human
cancer cells were grown were determined. The cells used were MCF7 breast
cancer cells and HCT80 colon cancer cells. The presumed SCM-factor
molecules from the supernatants were isolated and purified to homogeneity
using the combined procedures of Examples 1, 3, and 5 as described above
for the purification of SCM factor from blood plasma of cancer patients.
The sequence analyses on 8-picomolar amounts of the factor isolated from
the supernatant culture medium from human MCF7 breast cancer cells
identified the first 16 NH.sub.2 -terminal residues as:
M-I-P-P-E-V-X-F-N-K-P-F-(V-I-F-M). The last four residues gave a weak
signal. With two relatively conservative substitutions, methionine for
valine in position 1 and isoleucine for leucine in position 15, 15 out of
16 of the amino acids of this segment are identical with the majority
sequence of the SCM factor isolated from plasma of patients with breast
cancer in Example 14. At position 1, methionine was also found in the
purified preparation from blood plasma as a less-frequent alternative to
valine.
Similarly, 5-picomole amounts of purified SCM factor from the supernatant
culture medium of the culture of HCT-80 colon cancer cells were sequenced.
Six amino-terminal amino acids were determined: M-I-P-P-X-V. Five out of
six of these amino acids are identical to those determined on the SCM
preparation purified from blood plasma of patients with colon cancer. The
amino acid in position 4, denoted as X, could not be determined due to the
weak signal.
Example 25
Reactivity of SCM Factor Secreted from Human Cancer Cells In Culture with
Anti-SCM-factor Antibodies
The SCM factors secreted from human cancer cells in culture whose amino
acid sequences were presented in Example 24 also reacted with the anti-SCM
antibody of Example 20. A variation on the ELISA assay of Example 21 was
used. In this version of the ELISA assay, the assay was performed directly
on the eluate from the RP-HPLC purification step that remained adsorbed to
the Eppendorf.TM. collection tubes after loading of the bulk of the
eluates onto the sequenator disk. No other SCM factor was added, and there
was no additional sample added to the assay. This version of the SCM ELISA
assay is noncompetitive; the larger is the quantity of SCM factor adsorbed
to the Eppendorf.TM. tubes, the higher is the measured A.sub.405. The
results, shown in Table 15, clearly indicate the presence of material able
to react with anti-SCM antibody in these fractions.
TABLE 15
______________________________________
ELISA ASSAYS ON SCM FACTOR IN RP-HPLC
ELUATES PURIFIED FROM CULTURE MEDIA
OF CANCER CELLS
ELISA A.sub.405
Sample Signal/Background
Origin of Eluate Number Ratio.sup.a
______________________________________
MCF7 Breast Cancer Cells
1 43
MCF7 Breast Cancer Cells
2 17
MCF7 Breast Cancer Cells
3 71
HCT80 Colon Cancer Cells
1 34
HCT80 Colon Cancer Cells
2 12
______________________________________
.sup.a Background is ELISA A.sub.405 in tubes without adsorbed SCM factor
Example 26
Detection of SCM Factor in Human Cancer Cells In Culture by ELISA Assay
Human cancer cells in culture were directly shown to contain SCM-factor
molecules by antibody reactivity. Washed cells from monolayered cultures
of several human cancer cells: MCF7 breast cancer cells; T1080
fibrosarcoma cells; A2780 ovarian cancer cells; and HCT80 colon cancer
cells, were assayed directly by the noncompetitive ELISA assay procedure
of Example 25. The data is presented in Table 16. The calculated ELISA
absorbance ratios (i.e., the absorbance in the presence of anti-SCM
antibody divided by the absorbance in the absence of anti-SCM antibody,
which are a relative measure of the amounts of SCM factor per
4.times.10.sup.6 cells) showed that different cancer cell lines produced,
under identical conditions, different amounts of SCM factor.
Treatment of cultured cancer cells with the protein synthesis inhibitor
cycloheximide indicated that inhibition of protein synthesis decreased the
concentration of SCM factor associated with the cultured cancer cells. The
decrease was 25.3% for MCF7 breast cancer cells and 34% for T1080
fibrosarcoma cells. This data is presented in Table 17.
TABLE 16
______________________________________
SCM FACTOR IN HUMAN CANCER CELLS IN
CULTURE AS DETECTED BY ELISA ASSAYS
USING ANTI-SCM FACTOR ANTIBODY
Human Cancer Cell Line
(4 .times. 10.sup.6 cells)
ELISA A.sub.405 Ratio.sup.a
______________________________________
MCF7 Breast Cancer Cells
6.0
MCF7 Breast Cancer Cells
10.0
MCF7 Breast Cancer Cells
7.0
T1080 Fibrosarcoma Cells
6.5
A2780 Ovary Cancer Cells
4.6
HCT80 Colon Cancer Cells
3.0
______________________________________
##STR12##
TABLE 17
__________________________________________________________________________
EFFECT OF CYCLOHEXIMIDE ON SCM FACTOR
SYNTHESIS IN HUMAN CANCER CELLS
IN CULTURE AS DETECTED BY ELISA
ASSAYS USING ANTI-SCM FACTOR ANTIBODY
Cancer Cell Line
Cycloheximide
Incubation
Corrected
Corrected A.sub.405
(4 .times. 10.sup.6 cells)
(.mu.g/10.sup.6 cells)
(hrs) A.sub.405.sup.a
As % of Control
__________________________________________________________________________
MCF7 Breast Cancer
0 0 2.0716
100.0
MCF7 Breast Cancer
20 3 1.8893
91.2
MCF7 Breast Cancer
0 0 0.9654
100.0
MCF7 Breast Cancer
50 16 0.7217
74.7
T1080 Fibrosarcoma
0 0 1.5060
100.0
T1080 Fibrosarcoma
50 16 0.9940
66.0
__________________________________________________________________________
.sup.a Corrected A.sub.405 = (ELISA A.sub.405 in presence of cells)
(ELISA A.sub.405 in absence of cells)
Example 27
Effect of SCM Factor on DNA Synthesis
The effect of SCM factor on DNA synthesis was studied on normal rat
hepatocytes grown in culture. Details of the procedure were as described
in I. Hayashi & B. I. Carr, "DNA Synthesis in Rat Hepatocytes: Inhibition
by a Platelet Factor and Stimulation by an Endogenous Factor," J. Cell.
Physiol. 125, 82 (1985). Briefly, hepatocytes were obtained by the
high-pressure collagenase perfusion technique from male rat livers. The
cells were plated at 3.times.10.sup.5 cells per 35-mm tissue cultured dish
in Dulbecco's modified Eagle's growth medium (DME) supplemented with 10%
calf serum. Three hours after plating the cells, the medium was changed to
serum-free DME to which 10 ng/ml of epidermal growth factor (EGF) was
added to trigger DNA synthesis. To aliquots of the cell suspension, SCM
factor was added in concentrations of 1000 ng/ml, 100 ng/ml, 10 ng/ml, 1.0
ng/ml, and 0.1 ng/ml. The culture medium was changed daily and the culture
plates were kept at 37.degree. C. in 5% CO.sub.2 -air atmosphere.
About 72 hours after plating, 5 .mu.Ci/ml of tritiated thymidine was added
to each dish for about 6 to 8 hours. DNA synthesis was measured on cells
scraped off the tissue plates with rubber policemen into glass tubes and
collected by centrifugation. After washing once with PBS, 2 ml of cold 10%
trichloroacetic acid (TCA) was added to each tube and the cells were kept
at 4.degree. C. for one hour. After an additional wash with 10% TCA, the
cells were collected by centrifugation and hydrolyzed in 1 ml of 0.5 N
NaOH at 37.degree. C. overnight. Aliquots of these samples were used for
protein assay using colorimetric measurement at 595 nm after staining with
the protein-specific dye Coomassie brilliant blue. The remainder of the
samples were used for measurement of tritiated thymidine incorporation.
For this, samples were precipitated by addition of 0.25 ml of 50% TCA;
after 10 minutes on ice, they were passed through Whatman GF/C filters and
dried. The uptake of tritiated thymidine into acid-precipitable material
was counted in a Beckman scintillation counter. The results are presented
as counts per minute incorporated per milligram of cellular protein in
Table 18. The dose effect was highest at the lowest dose of the SCM
factor, i.e., at 0.1 ng/ml, and decreased at higher doses to a slight
inhibition of DNA synthesis (13 percent) at 1000 ng/ml. Since the
enhancement effect of SCM factor on DNA synthesis was assayed in this
system in addition to the effect of EGF as a promoter of cell growth and
DNA synthesis, the inhibitory effect at the highest dose of the SCM factor
could be the reaction to excessive stimulation in the presence of both the
SCM factor and EGF.
TABLE 18
______________________________________
EFFECT OF SCM FACTOR ON DNA SYNTHESIS
OF RAT HEPATOCYTES
SCM Factor Dose
Relative DNA Synthesis
(ng/ml) Per .mu.g DNA (%).sup.a
______________________________________
1000 86.5
100 134.5
10 171.5
1 184.0
0.1 192.4
______________________________________
##STR13##
Data was corrected for quenching caused by varying protein content.
Example 28
Effect of SCM Factor on Inhibition of Serine Proteases by .alpha.-1-PI
Protease Inhibitor
A protease activity using casein-resorufin as a protease substrate was
performed to determine the effect of SCM factor on inhibition of the
serine proteases trypsin, elastase, and cathepsin G by the serine protease
inhibitor .alpha.-1-PI. The protocol provided by the manufacturer,
Boehringer-Mannheim Biochemica, was followed to assay protease activity.
Table 19 shows the results when trypsin was used, Table 20 shows the
results when elastase was used, and Table 21 shows the results when
cathepsin G was used. In each case, SCM factor by itself did not affect
the proteolytic activity. However, when SCM factor was added before
.alpha.-1-PI or simultaneously in a mixture with .alpha.-1-PI, it
prevented the inhibition of trypsin by .alpha.-1-PI. The degree of this
effect depended on the quantity of SCM factor added. In contrast, as can
be seen in Table 19, when .alpha.-1-PI was allowed to react first with
trypsin, subsequent addition of SCM factor did not reverse the inhibition
of trypsin by .alpha.-1-PI. The active portion of the molecule that
prevents inhibition of the proteolytic enzymes by .alpha.-1-PI resides
within the first seven amino-terminal amino acid residues of the SCM
factor. This portion of the molecule is designated "fraction 6" or F6. The
effectiveness of equimolar amounts of the peptide F6, as compared to the
entire SCM factor molecule (Table 20) in preventing the inhibition of
elastase by .alpha.-1-PI is shown in Table 22.
TABLE 19
______________________________________
EFFECT OF SCM FACTOR ON INHIBITION OF
TRYPSIN ACTIVITY BY .alpha.-1-PI PROTEASE INHIBITOR
Trypsin Activity in
Casein-Resorufin
Assay as % of
Sequence of Addition
Untreated
of Reaction Components.sup.a
Control Enzyme
______________________________________
3 .mu.g Trypsin (control)
100.0
3 .mu.g Trypsin + 230 .mu.g SCM Factor
99.9
3 .mu.g Trypsin + 6.6 .mu.g .alpha.-1-PI
13.6
3 .mu.g Trypsin + 6.6 .mu.g -1-PI
14.0
(60 min inc.) + 230 .mu.g SCM Factor
3 .mu.g Trypsin + 21 .mu.g SCM Factor
15.6
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
3 .mu.g Trypsin + 60 .mu.g SCM Factor
40.0
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
3 .mu.g Trypsin + 100 .mu.g SCM Factor
62.5
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
3 .mu.g Trypsin + 150 .mu.g SCM Factor
97.7
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
3 .mu.g Trypsin + 230 .mu.g SCM Factor
101.7
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
6.6 .mu.g .alpha.-1-PI + 230 .mu.g SCM Factor
100.6
(10 min inc.) + 3 .mu.g Trypsin
______________________________________
.sup.a Total reaction volume is 300 .mu.L.
TABLE 20
______________________________________
EFFECT OF SCM FACTOR ON INHIBITION OF
ELASTASE ACTIVITY BY .alpha.-1-PI PROTEASE INHIBITOR
Trypsin Activity in
Casein-Resorufin
Assay as % of
Sequence of Addition Untreated
of Reaction Components.sup.a
Control Enzyme
______________________________________
3.2 .mu.g Elastase (control)
100.0
3.2 .mu.g Elastase + 230 .mu.g SCM Factor
100.0
3.2 .mu.g Elastase + 6.6 .mu.g .alpha.-1-PI
3.5
3.2 .mu.g Elastase + 10 .mu.g SCM Factor
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
6.4
3.2 .mu..mu.g Elastase + 30 g SCM Factor
7.0
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
3.2 .mu.g Elastase + 60 .mu.g SCM Factor
55.8
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
3.2 .mu.g Elastase + 100 .mu.g SCM Factor
71.1
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
3.2 .mu.g Elastase + 150 .mu.g SCM Factor
77.5
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
3.2 .mu.g Elastase + 230 .mu.g SCM Factor
90.1
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
______________________________________
.sup.a Total reaction volume is 300 .mu.L.
TABLE 21
______________________________________
EFFECT OF SCM FACTOR ON INHIBITION OF
CATHEPSIN G ACTIVITY BY .alpha.-1-PI
PROTEASE INHIBITOR
Cathepsin G
Activity in Casein-
Resorufin Assay as
Sequence of Addition of
% of Untreated
Reaction Components.sup.a
Control Enzyme
______________________________________
1 .mu.g Cathepsin G (control)
100.0
1 .mu.g Cathepsin G + 240 .mu.g SCM
97.0
Factor
1 .mu.g Cathepsin G + 6.6 .mu.g .alpha.-1-PI
20.0
1 .mu.g Cathepsin G + 240 .mu.g SCM
103.0
Factor (10 min inc.) + 6.6 .mu.g .alpha.-1-PI
______________________________________
.sup.a Total reaction volume is 300 .mu.L.
TABLE 22
______________________________________
EFFECT OF AMINO-TERMINAL PEPTIDE FRAGMENT
F6 (AMINO ACIDS 1-7) OF SCM FACTOR ON IN-
HIBITION OF ELASTASE ACTIVITY BY .alpha.-1-PI
PROTEASE INHIBITOR
Elastase Activity
in Casein-Resorufin
Assay as % of
Sequence of Addition of
Untreated Control
Reaction Components.sup.a
Enzyme
______________________________________
3.2 .mu.g Elastase (control)
100.0
3.2 .mu.g Elastase + 120 .mu.g F6
98.0
3.2 .mu.g Elastase + 6.6 .mu.g .alpha.-1-PI
3.4
3.2 .mu.g Elastase + 7.2 .mu.g F6
3.8
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
3.2 .mu.g Elastase + 21.6 .mu.g F6
9.8
(10 min. inc.) + 6.6 .mu.g .alpha.-1-PI
3.2 .mu.g Elastase + 30.0 .mu.g F6
34.2
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
3.2 .mu.g Elastase + 60.0 .mu.g F6
38.0
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
3.2 .mu.g Elastase + 120.0 .mu.g F6
96.3
(10 min inc.) + 6.6 .mu.g .alpha.-1-PI
______________________________________
.sup.a Total reaction volume is 300 .mu.L.
Example 29
Interaction of Protease Inhibitor .alpha.-1-PI With SCM-Factor Receptors
The protease inhibitor .alpha.-1-PI was shown to interact strongly with
SCM-factor receptors on SCM-responding lymphocytes from cancer patients.
This interaction was shown by the removal of the SCM-factor receptors from
such lymphocytes by washing after incubation with .alpha.-1-PI. This
caused reversion of the SCM response to that typical of lymphocytes from
donors free of cancer.
SCM-responding lymphocytes were isolated from blood samples of a patient
with malignant melanoma and from a normal, healthy donor. Half of the
lymphocyte suspension from each sample was incubated with 30 nanomoles of
.alpha.-1-PI per 6.times.10.sup.6 cells for 2.5 hours at 37.degree. C.;
the other half was retained as an untreated control. The cells were then
washed three times with PBS. Aliquots from both the untreated controls and
the .alpha.-1-PI-treated samples were challenged for 40 min with
phytohemagglutinin (PHA) or SCM factor. The SCM responses were measured by
changes in intracellular fluorescein fluorescence polarization as
described above under "Performance of the SCM Test." As can be seen in
Table 23, the control lymphocytes from the melanoma patients had a
RR.sub.SCM of 0.63, a typical value for lymphocytes from donors with
cancer. The same lymphocytes pre-incubated with .alpha.-1-PI had an
RR.sub.SCM of 1.8, a value typical of lymphocytes from healthy donors. By
contrast, incubation of lymphocytes from normal, healthy donors with
.alpha.-1-PI did not materially effect the responses of these cells in the
SCM test to PHA. The RR.sub.SCM was 1.74 in the absence of incubation and
1.68 after incubation.
TABLE 23
______________________________________
SCM-RESPONSE MODIFYING EFFECT OF .alpha.-1-PI
SCM Responses:
P Value as
Diagnosis of
in Vitro % of Control to:
Lymphocyte
Treatment of SCM
Donors Lymphocytes PHA Factor
RR.sub.SCM
______________________________________
Malignant
None 103.7 62.5 0.63
Melanoma 2.5 hr .alpha.-1-PI + 3X Wash
53.5 100.2 1.80
Healthy None 57.5 101.7 1.74
2.5 hr .alpha.-1-PI + 3X Wash
61.2 103.6 1.68
______________________________________
Example 30
Inhibition of SCM Factor Synthesis
As shown in Table 17 of Example 26, treatment of human cultured cancer
cells with cycloheximide, an inhibitor of protein synthesis, decreases the
amount of SCM factor produced by these cells. Similarly, ascorbic acid was
shown to inhibit the synthesis of SCM factor by MCF7 human breast cancer
cells in culture. The amounts of SCM factor per 7.times.10.sup.6 cells in
the presence or absence of 10.sup.-3 molar ascorbic acid after a 16-hour
incubation were measured by the noncompetitive ELISA procedure of Example
25. The results are shown in Table 24. The aliquot of cancer cells
incubated in the presence of ascorbate ions produced 43.9% less SCM factor
than untreated control cells. The observed inhibition of SCM factor
synthesis by ascorbic acid could be the result of decreased metabolic
activity of treated cancer cells since ascorbic acid was shown to
selectively induce in cancer cells the transition of mitochondria into the
idling, orthodox conformation, as described in L. Cercek & B. Cercek,
"Effects of Ascorbate Ions on Intracellular Fluorescein Emission
Polarization Spectra in Cancer and Normal Proliferating Cells," Cancer
Detection & Prevention 10, 1-20 (1987).
TABLE 24
______________________________________
EFFECT OF ASCORBATE IONS ON SCM FACTOR
SYNTHESIS IN CULTURED MCF7 HUMAN BREAST
CANCER CELLS
Corrected
Treatment During 16 hrs of
Corrected ELISA A.sub.405.sup.a as
Incubation at 37.degree. C.
ELISA A.sub.405.sup.a
% of Control
______________________________________
None (Control) 1.7448 100.0
1 .times. 10.sup.-3 M L-ascorbic acid,
0.9788 56.1
pH 7.1
______________________________________
.sup.a Corrected ELISA A.sub.405 = (ELISA A.sub.405) (Background
A.sub.405)
Example 31
Effect of Synthetic SCM Factor and Fragments Thereof on Natural
Cytotoxicity of Lymphocytes
The purified synthetic SCM factor molecule and peptides F1-F5, F7, and F8,
as described above in Example 16, were investigated to determine their
suppressive effect on the natural cytotoxicity of lymphocytes against
cancer cells, using the human myeloid cell line K 562 as target as
measured by the conventional .sup.51 Cr release method (Example 13).
In brief, the K 562 target cells grown in suspension in RPMI-FBS culture
medium were washed and labeled for 3 hours at 37.degree. C. with 100
.mu.Ci sodium [.sup.51 Cr]chromate and then washed 4 times in RPMI growth
medium. The effector lymphocytes used were isolated from heparinized
peripheral blood of normal healthy donors using either appropriate density
solutions to isolate the SCM-responding subpopulation of lymphocytes, as
described above under "Isolation of SCM-responding Lymphocytes," or the
conventional separation medium Histopaque 1077 for isolation of the total
population of peripheral blood lymphocytes (PBL). The effector cell to
target cell ratio used in these experiments was 40:1. Control samples
contained target cells only and were used to determine background isotope
release from the target cells. The test samples contained 0.2 mL aliquots
of labeled target cells to which 0.2 mL of effector lymphocytes was added;
the effector lymphocytes were either untreated or had been pretreated with
either the entire synthetic SCM factor or any of the fragments designated
F1 to F5, F7, or F8 for 2.5 hours at 37.degree. C. The tests were carries
out in triplicate. Cell suspensions were incubated for 18 hours at
37.degree. C. in an atmosphere of 95% air and 5% CO.sub.2. Samples were
then centrifuged for 10 minutes at 200.times.g. From each test sample
tube, a 0.2 mL aliquot of the supernatant was removed. Both the
supernatant and the remaining cell pellet were counted in a Beckman gamma
counter. The maximum possible isotope release was taken as the count
obtained by adding the counts for the supernatant and cell pellets. The
percentage of .sup.51 Cr release was determined for each of the triplicate
samples; using the mean value of .sup.51 Cr release, the percentage of
cytotoxicity was calculated according to the equation presented in Table
25.
As shown in the experiments whose results are given in Table 25, incubation
of SCM-responding lymphocytes and of PBL with 35 femtomoles of synthetic
SCM factor per lymphocyte decreased their natural killing efficiency or
cytotoxicity against K 562 cells by 97% to 99.9% as compared to
cytotoxicity of the untreated effector lymphocytes. This confirms the
results previously obtained with the natural SCM factor present in
ultrafiltrates of blood plasma from patients with cancer. The synthetic
SCM factor suppresses the cytotoxicity of both the SCM-responding
subpopulation of lymphocytes and the entire PBL population. As shown in
Table 25, the NK-suppressive effect of synthetic SCM factor is
irreversible and cannot be reversed by multiple washing of treated
lymphocytes.
TABLE 25
______________________________________
EFFECT OF SYNTHETIC SCM FACTOR ON
NATURAL LYMPHOCYTE CYTOTOXICITY
AGAINST K 562 MYELOID CELLS
Percent Cytotoxicity.sup.b :
Lymphocyte
Untreated Treated Treated & Washed
Fraction.sup.a
Lymphocytes Lymphocytes.sup.c
Lymphocytes
______________________________________
SCM-R 84.0 .+-. 3.0
0.1 .+-. 0.9
0.1 .+-. 0.9
SCM-R 72.0 .+-. 2.8
0.8 .+-. 0.7
--
SCM-R 45.6 .+-. 2.7
0.1 .+-. 0.8
--
PBL 77.8 .+-. 2.5
1.2 .+-. 1.0
--
PBL 72.7 .+-. 1.2
3.2 .+-. 0.7
3.0 .+-. 0.8
PBL 80.4 .+-. 1.8
2.6 .+-. 0.9
3.0 .+-. 0.8
______________________________________
.sup.a All lymphocytes were isolated from normal healthy donors. SCMR is
the subpopulation of peripheral blood lymphocytes isolated from density
gradient solutions, that yield the subpopulation which responds to
phytohaemagglutinin (PHA) in the SCM test. PBL is the total population of
peripheral blood lymphocytes isolated on Histopaque 1077 density solution
.sup.b Cytotoxicity was determined as in Example 13.
.sup.c Treated lymphocytes were treated with 35 femtomoles of synthetic
SCM factor per lymphocyte.
Values given are the mean of triplicate tests in each experiment. The
effector/target ratio was 40:1.
To establish which part of the SCM-factor molecule is responsible for this
NK-suppressive effect, we have treated the effector lymphocytes with
peptides representing different regions of the amino acid sequence of
synthetic SCM factor. As shown in Table 26, peptides that did not contain
the seven carboxyl-terminal amino acids had no effect on the natural
cytotoxicity of lymphocytes. However, peptides containing residues 14-29
(fragment F2) or 23-29 (fragment F8) are also inactive in suppressing
cytotoxicity. Therefore the NK-suppressive activity appears to require the
presence of amino acid residues 8-29 of the synthetic SCM molecule. This
region is different from the region of synthetic SCM factor responsible
for protection of protease from inhibition by .alpha.-I-PI, which resides
in the amino terminus of the molecule (Example 28). It is also more
extensive than the region of the SCM molecule responsible for SCM activity
(residues 14-22), although it overlaps that region (Example 17). For
example, peptide fragment F4, which extends from residue 14 to residue 22
and is fully active in the SCM test, does not cause any suppression of NK
activity.
TABLE 26
__________________________________________________________________________
DETERMINATION OF THE CYTOTOXICITY SUPPRESSIVE PORTION
OF THE SYNTHETIC SCM MOLECULE
Percent Cytotoxicity.sup.a :
Synthetic
Control
SCM Factor
F1 F2 F3 F4 F5 F7 F8
Untreated
(1-29).sup.b
(1-22)
(8-29)
(8-22)
(14-22)
(1-13)
(14-29)
(23-29)
__________________________________________________________________________
72.7 .+-. 1.2
3.2 .+-. 0.1
78.5 .+-. 1.0
14.9 .+-. 1.5
74.5 .+-. 1.5
76.2 .+-. 1.3
72.7 .+-. 1.3
-- --
80.4 .+-. 1.0
3.1 .+-. 0.8
81.0 .+-. 1.0
2.6 .+-. 1.7
80.5 .+-. 1.0
81.0 .+-. 1.5
79.8 .+-. 0.9
-- --
46.5 .+-. 1.5
0.1 .+-. 0.8
-- 2.4 .+-. 0.2
-- 48.6 .+-. 1.3
-- -- --
71.2 .+-. 1.5
-- -- -- -- -- -- 72.7 .+-. 1.0
71.7 .+-. 1.2
31.0 .+-. 2.0
-- -- -- -- -- -- -- 33.0 .+-. 1.8
__________________________________________________________________________
.sup.a Percent cytotoxicity was determined as in Example 13 using PBL.
Lymphocytes were incubated with 35 femtomoles of either the entire
synthetic SCM factor or peptides representing portions of it, for 2.5
hours at 37.degree. C.
.sup.b The numerals in parentheses indicate the numbers of the amino acid
residues included in the particular fragment of synthetic SCM factor.
Values given are the mean of triplicate tests in each experiment. The
effector/target ratio was 40:1.
ADVANTAGES OF THE PRESENT INVENTION
Both the isolated and purified SCM factors of the present invention and the
synthetic SCM factors and fragments of the present invention meet the
needs previously enumerated. In particular, they allow the assay of the
SCM response by the use of homogeneous challenging agents of defined
structure. The availability of such challenging agents eliminates the need
to use challenging agents partially purified from tissue extracts in the
SCM assay. Such partially purified challenging agents are non-homogeneous,
can vary from batch to batch in purity and potency, and can contain
contaminants that interfere with the SCM test. The challenging agents of
the present invention therefore give greater reliability and uniformity in
both clinical and research studies.
The knowledge of the complete amino acid sequences of such SCM factor
molecules allows their synthesis in quantity by either solid-phase peptide
synthesis techniques or, alternatively, by genetic engineering techniques
that can express the peptides in cells containing recombinant DNA.
Additionally, the availability of homogeneous and purified preparations of
SCM factors makes possible the preparation of antibodies specific to them.
The preparation of such antibodies allows the performance of immunoassays
for the detection and monitoring of small quantities of SCM factor in
vivo. This gives a new, noninvasive tool for the early detection of cancer
and the monitoring of the efficacy of cancer treatment. Such assays can,
for example, identify metastases before they would be otherwise
detectable. Such antibodies can also be used directly in treatment methods
to reduce the effect of SCM factors in vivo and enhance the resistance of
cancer patients to the disease.
In addition to assays of clinical importance, the availability of such
factors opens up new avenues for studying cancer. The ability to vary the
amino acid sequences of the SCM factors in known ways allows the
performance of structure-activity studies otherwise impossible. Also, the
ability to label such factors allows the isolation of receptors or other
molecules that interact with SCM factors in vivo. The pure, appropriately
labeled SCM factor can be used for detection of receptors for the SCM
factor on patients' lymphocytes. This can provide an alternative to the
SCM test.
Although the present invention has been described in considerable detail
with regard to certain preferred versions thereof, other versions are
possible. Therefore, the spirit and scope of the appended claims should
not be limited to the descriptions of the preferred versions contained
herein.
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